TW202404637A - Pharmaceutical compositions of anti-cd20/anti-cd3 bispecific antibodies and methods of use - Google Patents

Abstract

The present invention relates to pharmaceutical compositions of anti-CD20/anti-CD3 bispecific antibodies and methods of using the same.

Description

Pharmaceutical compositions and methods of use of anti-CD20/anti-CD3 bispecific antibodies

The present invention relates to pharmaceutical compositions of anti-CD20/anti-CD3 bispecific antibodies and methods of use thereof.

One of the major challenges in developing biotech therapeutics is protein stability, which must be maintained throughout the multiple process steps involved in bringing these biotech therapeutics to market. Furthermore, protein stability must be maintained during storage and during administration to patients. The therapeutic antibodies may be formulated in an aqueous vehicle for administration to an individual, for example, by intravenous or subcutaneous administration. During the storage, handling and administration of such pharmaceutical compositions, it is necessary to mitigate the loss of therapeutic antibodies, which may occur through degradation and surface adsorption, such as protein adsorption to filters, storage tanks, tubes, syringes, intravenous Surface of intravenous bags and other containers. Low- and high-concentration formulations have their own challenges during research and development and manufacturing. For example, low concentrations are greatly affected by surface adsorption, while high concentrations can exhibit high viscosity.

In cases where pharmaceutical compositions contain relatively low concentrations of therapeutic proteins, these factors can significantly increase protein loss, resulting in reduced therapeutic efficacy of the pharmaceutical composition.

Therefore, there is a need in the field to develop pharmaceutical formulations in which anti-CD20/anti-CD3 bispecific antibodies (e.g., low-dose anti-CD20/anti-CD3 bispecific antibodies, e.g., low-dose anti-CD20/anti-CD3 T cell-engaging bispecific antibodies Antibodies, such as glofitamab, are stable and cannot be lost by adsorption.

The present invention relates to pharmaceutical compositions of anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 T cell engaging bispecific antibodies (TCB), such as gaffetuzumab, RO7082859 or RG6026) and methods of use thereof . The disclosed compositions and related methods solve the problem of delivering anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) formulated at low concentrations, ensuring that during storage and Patients receive the desired dose of an anti-CD20/anti-CD3 bispecific antibody (eg, anti-CD20/anti-CD3 TCB, eg, gaffetuzumab) with little protein loss during administration.

In one aspect, the invention features a liquid pharmaceutical composition comprising: Approximately 1 to 25 mg/ml of anti-CD20/anti-CD3 bispecific antibody; About 10 to 50 mM buffer; Approximately ≥ 200 mM tonicity agent; approximately 0 to 15 mM methionine; and Approximately ≥ 0.2 mg/ml surfactant; pH in the range of about 5.0 to about 6.0, Among them, anti-CD20/anti-CD3 bispecific antibodies include a) At least one antigen-binding domain that specifically binds to CD20, which includes Heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3, which includes Heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) concentration is in the range of about 1 to 5 mg/ml. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) concentration is in the range of about 0.9-1.1 mg/ml. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) concentration is about 1 mg/ml.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises a) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and b) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises a) A first Fab molecule that specifically binds to CD3, specifically CD3 epsilon; and in which the variable domains VL and VH of the Fab light chain and Fab heavy chain replace each other; b) The second Fab and the third Fab molecule, which specifically bind to CD20, wherein in the constant domain CL of the second Fab and the third Fab molecule, the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced (according to Kabat numbering) by lysine (K) or arginine (R), specifically arginine (R), and where in In the constant domain CH1 of the second and third Fab molecules, the amino acid at position 147 is replaced by glutamic acid (E) (EU number), and the amino acid at position 213 is replaced by glutamic acid (E ) replaces (EU number); and c) Fc domain, which consists of the first unit and the second unit that can be stably associated.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody is gaffetuzumab.

In one embodiment, the buffer is histidine buffer, optionally histidine HCl buffer. In one embodiment, the buffer is at a concentration of about 15 to 25 mM. In one embodiment, the buffer is at a concentration of about 20 mM. In one embodiment, the buffer provides a pH of about 5.2 to about 5.8.

In one embodiment, the tonicity agent is selected from the group of salts, sugars and amino acids. In one embodiment, the tonicity agent is sucrose or sodium chloride. In one embodiment, the tonicity agent is sucrose at a concentration of about 200 mM or greater. In one embodiment, the tonicity agent is sucrose at a concentration of about 200 mM to 280 mM. In one embodiment, the tonicity agent is sucrose at a concentration of about 240 mM.

In one embodiment, methionine is at a concentration of about 5 to 15 mM.

In one embodiment, methionine is at a concentration of about 10 mM. In one embodiment, the surfactant is at a concentration of about 0.2 to 0.8 mg/ml. In one embodiment, the surfactant is polysorbate 20 or poloxamer 188. In one embodiment, the surfactant is polysorbate 20 at a concentration of 0.2 to 0.8 mg/ml. In one embodiment, the surfactant is polysorbate 20 at a concentration of about 0.5 mg/ml.

In one embodiment, the liquid pharmaceutical composition contains: Approximately 1 to 5 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5 to about 6.

In one embodiment, the liquid pharmaceutical composition contains: Approximately 1 mg/ml of greffitumab; Approximately 20 mM histamine buffer; Approximately 240 mM sucrose; Approximately 10 mM methionine; and Approximately 0.5 mg/ml of PS20 The pH is about 5.5.

In one embodiment, the present invention provides the use of the liquid pharmaceutical composition of any one of the aforementioned aspects and embodiments for preparing a medicament for treating cell proliferative disorders.

In another aspect, the invention features a pharmaceutical composition of any one of the preceding aspects and embodiments for treating or delaying the progression of a cell proliferative disorder in an individual in need thereof.

In another aspect, the invention features a pharmaceutical composition of any one of the preceding aspects and embodiments for treating or delaying the progression of a cell proliferative disorder in an individual in need thereof, comprising administering to the individual An effective amount of the composition of any one of the aforementioned aspects and embodiments.

In specific embodiments, the cell proliferative disorder is cancer.

Another aspect of the invention relates to the invention as described herein.

Embodiments may be combined unless the context clearly dictates otherwise. The embodiments may apply to various aspects of the invention unless the context clearly dictates otherwise.

Specific embodiments of the invention will become apparent from the following more detailed description of certain preferred embodiments and claims.

The present invention relates to pharmaceutical compositions of anti-CD20/anti-CD3 bispecific antibodies and methods of use thereof. The disclosed compositions and related methods solve the problem of delivering anti-CD20/anti-CD3 bispecific antibodies formulated at low concentrations, ensuring that patients receive the intended dose of anti-CD20 with virtually no protein loss during storage and administration. /anti-CD3 bispecific antibody. I. General technology

Unless otherwise indicated, the practice of the invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the scope of the art. These techniques are fully described in documents such as: "Molecular Cloning: Laboratory Manual, Second Edition" (Sambrook et al., 1989); "Oligonucleotide Synthesis" ( "Oligonucleotide Synthesis", edited by MJGait, 1984); "Animal Cell Culture" (edited by RI Freshney, 1987); "Methods in Enzymology", Academic Press Press, Inc.); "Current Protocols in Molecular Biology" (edited by FM Ausubel et al., 1987, and regularly updated); "PCR: The Polymerase Chain Reaction"("PCR: The "Polymerase Chain Reaction", edited by Mullis et al., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual"("Phage Display: A Laboratory Manual”, Barbas et al., 2001). II.Definition _

Definitions Unless otherwise defined below, the terms used herein are those of ordinary use in the art.

Unless otherwise stated, the term "cluster of differentiation 20" or "CD20" as used herein refers to any native CD20 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). CD20 (also known as B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the human protein is described in UniProt database accession number P11836) has a molecular weight of approximately 35 kD and exhibits Hydrophobic transmembrane proteins on pre-B and mature B lymphocytes (Valentine, MA et al., J. Biol. Chem . 264 (1989) 11282-11287; Tedder, TF, et al., Proc. Natl. Acad. Sci. USA .85 (1988) 208-212; Stamenkovic, I., et al., J. Exp. Med . 167 (1988) 1975-1980; Einfeld, DA, et al., EMBO J.7 (1988) 711- 717; Tedder, TF, et al., J. Immunol . 142 (1989) 2560-2568). The corresponding human gene is transmembrane domain 4, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this nascent protein family are characterized by shared structural features and similar intron/exon splicing boundaries and display unique expression patterns between hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in B cell development and differentiation into plasma cells. This family member is located at 11q12, in a cluster of family members. The term covers "full-length" unprocessed CD20 as well as any form of CD20 produced by processing in the cell. The term also encompasses natural CD20 variants, such as splice variants or allelic variants. Alternative splicing of this gene results in two transcript variants encoding the same protein. In one embodiment, CD20 is human CD20.

The terms "anti-CD20 antibody" and "antibody that binds CD20" refer to antibodies that are capable of binding CD20 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting CD20. In one embodiment, the anti-CD20 antibody binds to an unrelated, non-CD20 protein to a degree that is about 10% less than the antibody binds to CD20, as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, the antibody that binds CD20 has a dissociation constant (K _D ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 ^-8 M or less, for example, 10 ^-8 M to 10 ^-13 M, for example, 10 ^-9 M to 10 ^-13 M). In certain embodiments, anti-CD20 antibodies bind to epitopes of CD20 that are conserved in CD20 across species.

"Type II anti-CD20 antibody" refers to an anti-CD20 antibody that has the binding properties and biological activity of type II anti-CD20 antibodies, such as Cragg et al., Blood 103 (2004) 2738-2743; Cragg et al., Blood 101 ( 2003) 1045-1052; Klein et al., mAbs 5 (2013), 22-33, and summarized in Table 1 below.

Table 1. Characteristics of Type I and Type II anti -CD20 antibodies Type I anti -CD20 antibodies Type II anti -CD20 antibodies Binds to class I CD20 epitope Binds to class II CD20 epitope Localizes CD20 to lipid membrane rafts Does not localize CD20 to lipid membrane rafts High CDC* Low CDC* ADCC activity* ADCC activity* Fully able to bind to B cells Binding ability to B cells is approximately halved weak homotypic aggregation Homotypic aggregation Low cell death induction Strong cell death induction * If it is IgG ₁ isotype

Examples of type II anti-CD20 antibodies include, for example, obinutuzumab (GA101), tositumumab (B1), humanized B-Ly1 antibody IgG1 (as disclosed in WO 2005/044859) combined humanized IgG1 antibody), 11B8 IgG1 (as disclosed in WO 2004/035607) and AT80 IgG1.

Examples of Type I anti-CD20 antibodies include, for example, rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab ( ocrelizumab), PRO131921, ublituximab, HI47 IgG3 (ECACC, fusion tumor), 2C6 IgG1 (as disclosed in WO 2005/103081), 2F2 IgG1 (as disclosed in WO 2004/035607 and WO 2005/103081 as disclosed) and 2H7 IgG1 (as disclosed in WO 2004/056312).

Unless otherwise stated, "CD3" refers to any native CD3 derived from any vertebrate, including mammals, such as primates (e.g., humans), non-human primates (e.g., macaques) and rodents (such as mice and rats). The term covers "full-length" unprocessed CD3 as well as any form of CD3 produced by processing in the cell. The term also encompasses natural CD3 variants, such as splice variants or allelic variants. In one embodiment, CD3 is human CD3, particularly the ε subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. The amino acid sequence of Macaca fascicularis CD3ε is shown in NCBI GenBank No. BAB71849.1.

The terms "anti-CD20/anti-CD3 antibody", "anti-CD20/anti-CD3 bispecific antibody" and "bispecific antibody that binds CD20 and CD3" refer to the ability to bind both CD20 and CD3 with sufficient affinity such that the Antibodies can be used as bispecific antibodies as diagnostic and/or therapeutic agents targeting CD20 and/or CD3. In one embodiment, the bispecific antibody that binds CD20 and CD3 binds to unrelated, non-CD3 proteins and/or non-CD20 proteins to a degree that is less than about 10% of the binding of the antibody to CD3 and/or CD20, such as, e.g. Measured by radioimmunoassay (RIA). In certain embodiments, the anti-CD20/anti-CD3 bispecific antibody binding to each of CD20 and/or CD3 has a dissociation constant ( _KD ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (eg 10 ^-8 M or less, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 to 10 ^-13 M). In certain embodiments, bispecific antibodies that bind CD20 and CD3 bind to a CD3 epitope that is conserved between CD3s from different species and/or a CD20 epitope that is conserved between CD20s from different species . An example of an anti-CD20/anti-CD3 bispecific antibody is gaffetuzumab (WHO Drug Information (International Nonproprietary Name), Proposed INN: List 83, 2020, Volume 34, Issue 1, Page 39, Also known as anti-CD20/anti-CD3 T-cell engaging bispecific antibody (TCB), CD20-TCB, RO7082859, or RG6026; CAS #: 2229047-91-8).

The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to obtain the final construct, provided that the final construct has the desired properties, such as reduced binding to Fc receptors. Deletions and insertions of amino acid sequences include deletions and insertions of the amino and/or carboxyl termini of amino acids. Specific amino acid mutations lead to amino acid substitutions. In order to change the binding characteristics of, for example, the Fc region, non-conservative amino acid substitution is particularly preferred, that is, one amino acid is replaced by another amino acid with different structure and/or chemical properties. Amino acid substitutions include non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine acid, homoserine, 5-hydroxylysine) replacement. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is expected that methods other than genetic engineering, such as chemical modification, to alter the side chain groups of amino acids may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, the substitution of proline to glycine at position 329 of the Fc region can be represented by 329G, G329, G ₃₂₉ , P329G or Pro329Gly.

"Affinity" refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (eg, a receptor) and its binding partner (eg, a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, receptor and ligand). The affinity of a molecule X for its partner Y can usually be expressed by the dissociation constant (K _D ), which is the ratio of the dissociation rate constant and the association rate constant (k _off and _kon respectively). Therefore, equivalent affinities can include different rate constants as long as the rate constant ratio remains the same. Affinity can be measured by established methods known in the art. A specific method used to determine affinity is surface plasmon resonance (SPR).

An "affinity matured" antibody is an antibody that has one or more changes in one or more highly variable regions (HVRs) that cause the antibody to react differently to the antigen compared to a parent antibody that does not have such changes. Improvement of affinity.

The term "antigen binding moiety" as used herein refers to a polypeptide molecule that specifically binds to an antigenic epitope. In one embodiment, the antigen-binding moiety is capable of directing the entity to which it is attached (e.g., a cytokine or a second antigen-binding moiety) to a target site, e.g., to a specific type of tumor cell bearing an epitope. or tumor stroma. Antigen-binding portions include antibodies and fragments thereof as further defined herein. Preferred antigen-binding portions include the antigen-binding domain of an antibody, which includes an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding portion may comprise an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of five isotypes: alpha, delta, epsilon, gamma, or mu. Useful light chain constant regions include either of two isotypes: kappa and lambda.

"Binds,""specificallybinds" or "specific for" means that binding is selective for the antigen and distinguishes undesired or nonspecific interactions. The ability of the antigen-binding moiety to bind to a specific epitope can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (e.g., analyzed on a BIACORE® instrument) ( Liljeblad et al., Glyco J. 17, 323-329 (2000)) and the traditional binding assay (Heeley, E 28, 217-229 (2002)). In one embodiment, the antigen-binding portion binds the unrelated protein to an extent that is less than about 10% of the antigen-binding portion bound by the antigen-binding portion, eg, as determined by SPR. In certain embodiments, the dissociation constant (K _D ) of the antigen-binding moiety that binds to the antigen or the antigen-binding molecule comprising the antigen-binding moiety is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (eg, 10 ^-8 M or less, eg, 10 ^-8 M to 10 ^-13 M, eg, 10 ^-9 M to 10 ^-13 M).

"Reduced binding," e.g., reduced binding to an Fc receptor, refers to a decrease in the affinity of the respective interaction, e.g., as measured by SPR. For the sake of clarity, the term also includes reducing the affinity to zero (or below the detection limit of the analytical method), that is, the interaction is completely abolished. In contrast, "increased binding" refers to an increase in the binding affinity of the respective interaction.

As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic epitope. Examples of antigen-binding molecules are immunoglobulins and their derivatives (eg, fragments).

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to the formation of the antigen-binding moiety-antigen on the polypeptide macromolecule to which the antigen-binding moiety binds The site of the complex (e.g., a continuous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids). For example, useful antigenic determinants may be found on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, are not present in serum and/or are present in the extracellular matrix (ECM) middle. Unless otherwise stated, a protein referred to herein as an antigen (e.g., CD3) may be any naturally occurring form of the protein from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents. Animals (such as mice and rats). In specific embodiments, the antigen is a human protein. Where reference is made herein to a specific protein, the term encompasses "full-length," unprocessed protein and any protein form resulting from processing in a cell. The term also encompasses naturally occurring protein variants, such as splice variants or allele variants. An example human protein that can be used as an antigen is CD3, specifically the epsilon subunit of CD3 (see UniProt number P07766 (ed. 130), NCBI RefSeq number NP_000724.1, which is for human sequences; or UniProt number Q95LI5 ( 49th edition), NCBI GenBank number BAB71849.1, this is for macaque [Macaca fascicularis] sequence). In certain embodiments, T cell activating bispecific antigen-binding molecules disclosed herein bind to epitopes of CD3 or target cell antigens that are conserved between CD3 or target cell antigens from different species.

As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not indicate a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain" or any other term used to refer to two or more amino acid chains, And "polypeptide" may be used instead of or interchangeably with any of these terms. The term "polypeptide" also refers to the products of post-expression modifications of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization via known protecting/blocking groups, proteolysis or non- Naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including through chemical synthesis. Polypeptides of the invention may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more , 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a definite three-dimensional structure, although they do not necessarily have such a structure. Polypeptides that have a definite three-dimensional structure are called folded, whereas polypeptides that do not have a definite three-dimensional structure but can adopt a large number of different configurations are called unfolded.

An "isolated" polypeptide or variant or derivative thereof refers to a polypeptide that is not found in its natural environment. No specific level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. For the purposes of this invention, recombinantly produced antibodies and proteins expressed in host cells are considered isolated, as native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, when comparing the sequences and introducing After divergence (if necessary), the maximum percent sequence identity is achieved and any retained substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN® (DNASTAR ®) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment over the entire length of the sequences being compared. However, for the purposes of this article, % amino acid sequence identity values were generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Jiannan Deke Corporation, and the original code is filed with the User Documentation in the U.S. Copyright Office, Washington, DC 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from JNANDK Corporation, South San Francisco, California, or can be compiled from source code. ALIGN-2 programs should be compiled for use on UNIX® operating systems (including digital UNIX® V4.0D). All sequence comparison parameters were set by the ALIGN-2 program and were unchanged. In the case of amino acid sequence comparison using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be expressed as A given amino acid sequence A, which has or contains a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where It should be understood that in the case where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A and B will not be equal to the % amino acid sequence identity of B and A. sex. Unless otherwise specifically stated, all % amino acid sequence identity values used in this article were obtained using the ALIGN-2 computer program as described in the previous paragraph. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit Antigen binding activity is expected.

The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to that of a native antibody or that has a heavy chain containing an Fc region as defined herein.

"Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies, linear antibodies, single chain antibody molecules (eg, scFv), and multispecifics formed from antigen fragments antibody. The term "antibody fragment" as used herein also encompasses single domain antibodies.

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, IgG immunoglobulins are heterotetrameric glycoproteins of about 150,000 Daltons composed of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain domains. chain constant region. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain; followed by a light chain constant (CL) domain, also known as Light chain constant region. The heavy chains of immunoglobulins can be classified into one of five categories, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further classified. Divided into subclasses such as γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ), and α ₂ (IgA ₂ ). Based on the amino acid sequence of their constant domains, immunoglobulin light chains can be classified into one of two types, termed kappa (κ) and lambda (λ). Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via the immunoglobulin hinge region.

The term "antigen binding domain" refers to the portion of an antibody that contains part or all of a region that specifically binds and is complementary to an antigen. The antigen-binding domain may be provided, for example, by one or more antibody variable domains (also known as antibody variable regions). Preferably, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL respectively) usually have similar structures, and each domain contains four conserved framework regions (FR) and three highly variable regions (HVR) . See, for example, Kindt et al., Kuby Immunology , 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

"Human antibody" is an antibody having an amino acid sequence corresponding to that produced by humans or human cells or from non-human sources utilizing human antibody repertoire or other human antibody coding sequences. Amino acid sequence of the derived antibody. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen binding residues.

"Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will include substantially all of at least one (and typically two) variant domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to non-human antibodies, and the like, and all or Virtually all FRs correspond to those of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

As used herein, the term "highly variable region" or "HVR" refers to each region of an antibody variable domain that is highly variable in sequence (a "complementarity determining region" or "CDR") and/or or form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen contacts"). Typically, antibodies contain six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). As used herein, exemplary HVRs include: (a) Highly variable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1) , 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDR exists at amino acid residue 24- 34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest , 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contacts occur at amino acid residues 27c-36 (L1), 46-55 (L2), 89 -96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and ( d) Combinations of (a), (b) and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 ( L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise stated, HVR residues and other residues in variable domains (e.g., FR residues) are referred to herein according to Kabat et al. (Same as above) Number.

"Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain usually consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The "human consensus skeleton" is a skeleton that represents the most common amino acid residues in a series of human immunoglobulin VL or VH skeleton sequences. Typically, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Typically, subgroups of sequences are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, subgroups such as Kabat Subgroup κ I described in the above literature by et al. In one embodiment, for VH, subgroups such as Kabat Subgroup III described in the above literature by et al.

"Acceptor human framework" for the purposes herein is an amine derived from a human immunoglobulin framework or a human consensus framework, including a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework, as defined below The framework of the amino acid sequence. A recipient human scaffold "derived from" a human immunoglobulin scaffold or a human consensus scaffold may contain the same amino acid sequence as these, or it may contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or more less, or 2 or less. In some embodiments, the VL receptor human scaffold is identical in sequence to a VL human immunoglobulin scaffold sequence or a human consensus scaffold sequence.

The "class" of an antibody refers to the constant domain or type of constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of these classes can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

As used herein, the term IgG "isotype" or "subtype" refers to any subtype of an immunoglobulin defined by the chemical and antigenic properties of its constant region.

The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two amino acids at the C-terminus of the heavy chain. Thus, antibodies produced by a host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or may include a cleavage variant of a full-length heavy chain (also referred to herein as a "cleavage variant heavy chain"). This may be due to the fact that the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU number). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise stated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index), such as Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, ie, the polypeptide that contains the C-terminal constant region of the immunoglobulin heavy chain that is capable of stabilizing self-association. For example, the subunits of the IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

"Modifications that promote the association of the first unit and the second unit of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunit that reduce or prevent polypeptides containing the Fc domain subunit Association with the same polypeptide forms homodimers. As used herein, modifications that promote association particularly include separate modifications to each of the two Fc domain subunits (i.e., the first unit and the second subunit of the Fc domain) that are desired to associate, wherein the modification complement each other to facilitate the association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both Fc domain subunits to render them sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which is further fused to the assembly of each subunit (e.g., antigen-binding moiety) Parts may vary. In some embodiments, modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a specific embodiment, modifications that promote association include individual amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

An "activating Fc receptor" is an Fc receptor that, upon engagement with the Fc region of an antibody, initiates signaling events that stimulate the cell carrying the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

The term "effector function" when used in reference to an antibody refers to those biological activities attributed to the Fc region of the antibody, which vary with the antibody isotype. Examples of antibody utility functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, Immune complex uptake by antigen-presenting cells mediates antigen, cell surface receptor (eg, B cell receptor) downregulation, and B cell activation.

As used herein, the term "effector cells" refers to a population of lymphocytes that display on their surface receptors for effector moieties (e.g., cytokine receptors) and/or Fc receptors through which they bind effector moieties (e.g., cytokine receptors) , cytokines) and/or the Fc region of the antibody and contributes to the destruction of target cells, such as tumor cells. Effector cells may, for example, mediate cytotoxic or phagocytic responses. Effector cells include, but are not limited to, effector T cells such as CD8 ⁺ cytotoxic T cells, CD4 ⁺ helper T cells, γδ T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages/monocytes.

The terms "engineering" as used herein are deemed to include any manipulation of the peptide backbone or post-translational modification of naturally occurring or recombinant polypeptides or fragments thereof. Engineering involves modifying the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these approaches. "Engineering" (especially with the prefix "glycosyl") and the term "glycosylation engineering" include metabolic engineering of cellular glycosylation machinery, including genetic manipulation of oligosaccharide synthesis pathways, to achieve intracellular Manifested by changes in glycosylation of glycoproteins. In addition, glycosylation modification includes mutations and the response of the cellular environment to glycosylation. In one embodiment, glycosylation modification alters glycosyltransferase activity. In a specific embodiment, the modification alters glucosaminyltransferase activity and/or fucosyltransferase activity. Glycosylation engineering can be used to obtain "host cells with increased GnTIII activity" (e.g., that have been manipulated to express increased levels of one or more beta(1,4)-N-acetylglucosaminyltransferase enzymes). III (GnTIII) active polypeptide), “host cells with increased ManII activity” (e.g., host cells that have been manipulated to express increased levels of one or more polypeptides with alpha-mannosidase II (ManII) activity host cells), or "host cells with reduced alpha(1,6)-fucosyltransferase activity" (e.g., that have been manipulated to express reduced levels of alpha(1,6)-fucosyltransferase host cell).

The terms "host cell", "host cell strain" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including progeny cells of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny cells derived therefrom, regardless of the number of passages. The nucleic acid content of the daughter cells may not be exactly the same as that of the parent cells, but may contain mutations. Mutated progeny cells having the same function or biological activity as that screened or selected in the original transformed cell are included herein. A host cell is any type of cell system that can be used to produce proteins for use in the invention. In one embodiment, the host cell is engineered to allow the production of antibodies with modified oligosaccharides. In certain embodiments, the host cell has been manipulated to express increased amounts of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments, the host cell has been further manipulated to express increased amounts of one or more polypeptides having alpha-mannosidase II (ManII) activity. Host cells include cultured cells, for example, mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or fusion tumors Cells, yeast cells, insect cells and plant cells, to name a few, but also include cells contained in genetically modified animals, genetically modified plants or cultured plant or animal tissues.

As used herein, the term "polypeptide with GnTIII activity" refers to trimannose capable of catalyzing the addition of N-acetylglucosamine (GlcNAc) residues in β-1,4 linkages to N-linked oligosaccharides. Nuclear β-linked mannosides. This term includes expressions similar to, but not necessarily identical to, β(1,4)-N-acetylglucosaminyltransferase III (also known as β-1,4-mannosyl-glycoprotein 4-β-N - Enzymatic activity of acetylglucosaminyl-transferase (EC 2.4.1.144), according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), as measured in a specific bioassay and with or without dose dependence) fusion polypeptides. Where a dose dependence does exist, it need not be identical to that of GnTIII, but rather the dose dependence of a given activity will be substantially similar to that of GnTIII (i.e., the candidate polypeptide will exhibit greater activity relative to GnTIII The activity may be reduced by no more than about 25 times, preferably by no more than about ten times, and most preferably by no more than about three times. In certain embodiments, the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi-resident polypeptide. Specifically, the Golgi localization domain is that of mannosidase II or GnTI, most specifically that of mannosidase II. Alternatively, the Golgi localization domain is selected from the group consisting of: a localization domain for mannosidase I, a localization domain for GnTII, and a localization domain for α1,6 core fucosyltransferase. Methods of generating such fusion polypeptides and using them to generate antibodies with increased reactivity are disclosed in WO2004/065540, U.S. Provisional Patent Application No. 60/495,142, and U.S. Patent Application Publication No. 2004/0241817, which The entire contents of which are expressly incorporated herein by reference.

As used herein, the term "Horizontal localization domain" refers to the amino acid sequence of a homogeneous resident polypeptide that is responsible for anchoring the polypeptide to a position within the homogeneous complex. Typically, the localization domain contains the amine-terminal "tail" of the enzyme.

As used herein, the term "polypeptide with ManII activity" refers to the terminal 1,3- and 1,6-linked α-D-mannose in the branched GlcNAcMan ₅ GlcNAc ₂ mannose intermediate capable of catalyzing N-linked oligosaccharides. Polypeptides hydrolyzed from sugar residues. This term includes expression similar to, but not necessarily identical to, Golgi α-mannosidase II (also known as mannosyl oligosaccharide 1,3-1,6-α-mannosidase II (EC 3.2.1.114), according to Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells to which an antibody or fragment thereof that contains the Fc region specifically binds, usually via the N-terminal protein portion of the Fc region. As used herein, the term "increased/decreased ADCC" is defined as an increase in the number of target cells lysed by the ADCC mechanism defined above in a given time with a given concentration of antibody in the culture medium surrounding the target cells. / Decrease, and/or decrease / increase in the concentration of antibody in the culture medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time by the ADCC mechanism. The increase/decrease in ADCC is mediated relative to the same antibody (but not yet engineered) produced by the same type of host cell using the same standard production, purification, formulation and storage methods (methods known to those of ordinary skill in the art) Directed ADCC. For example, relative to the same antibody-mediated ADCC produced by the same type of unengineered host cell, engineered by the methods disclosed herein to have an altered glycosylation pattern (e.g., to express a glycosyltransferase , GnTIII or other glycosyltransferases), increased ADCC is mediated by antibodies produced by host cells.

"An antibody with increased antibody-dependent cell-mediated cytotoxicity (ADCC)" means an antibody as defined herein that has an increased/decreased ADCC as determined by any suitable method known to one of ordinary skill in the art . An acceptable in vitro ADCC assay is as follows: 1) The assay uses target cells known to express the target antigen recognized by the antigen-binding region of the antibody; 2) The assay uses randomly selected cells from Human peripheral blood mononuclear cells (PBMC) isolated from the blood of healthy donors were used as effector cells; 3) Implement the assay according to the following operation protocol: i) Use standard density centrifugation procedures to isolate PBMC, and use 5 × 10 ⁶ Cells/ml Suspended in RPMI cell culture medium; ii) Grow target cells by standard tissue culture methods, harvest from exponential growth phase, with a survival rate greater than 90%, wash in RPMI cell culture medium, and use 100 microCuries of ⁵¹ Cr label, wash twice with cell culture medium, and resuspend in cell culture medium at a density of 10 ⁵ cells/ml; iii) Transfer 100 μl of the above final target cell suspension to the center of a 96-well microtiter plate in each well; iv) Serially dilute the antibody from 4000 ng/ml to 0.04 ng/ml in cell culture medium and add 50 μl of the resulting antibody solution to the target cells in a 96-well microtiter plate in triplicate Test various antibody concentrations covering the entire concentration range mentioned above in duplicate; v) For the maximum release (MR) control, add 50 µl of 2% (v/v) to 3 additional wells of the plate containing labeled target cells. ) a nonionic detergent aqueous solution (Nonidet, Sigma, St. Louis) instead of the antibody solution (point iv above); vi) for the spontaneous release (SR) control, 3 additional cells containing labeled target cells in the plate In each well, add 50 μl of RPMI cell culture medium instead of the antibody solution (point iv above); vii) Then centrifuge the 96-well microplate at 50 × g for 1 minute and incubate at 4°C for 1 hour; viii) Add 50 μl of PBMC suspension (point i above) to each well to give an effector:target cell ratio of 25:1 and place the plate in an incubator with a 5% CO _atmosphere . Place at 37°C for 4 hours; ix) Collect the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter; x) According to the formula (ER-MR)/(MR- SR) × 100 Calculate the percent specific lysis for each antibody concentration, where ER is the mean radioactivity quantified against that antibody concentration (see point ix above) and MR is the quantified against the MR control (see point v above) The average radioactivity (see point ix above), and SR is the average radioactivity quantified by the SR control (see point vi above) (see point ix above); 4) "ADCC of increase/decrease" is defined as the Increase/decrease in the maximum percentage of specific lysis observed over the range of antibody concentrations tested, and/or decrease/increase in the antibody concentration required to achieve half of the maximum percentage of specific lysis observed over the range of antibody concentrations tested above . The increase/decrease in ADCC is relative to the same antibody (but not modified) produced by the same type of host cell using the same standard production, purification, formulation and storage methods (methods known to those of ordinary skill in the art) Mediated ADCC (measured using the assay described above).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except that, for example, they contain naturally occurring mutations or In addition to the possible variant antibodies generated during the production of monoclonal antibody preparations, such variants usually exist in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes (epitopes), monoclonal antibody preparations have each monoclonal antibody system directed against a single epitope on the antigen. Accordingly, the modifier "monoclonal" indicates that the characteristics of the antibody were obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies intended for use in accordance with the invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and the use of gene transgenes containing all or part of the human immunoglobulin locus. Methods for breeding animals are described herein, as well as other exemplary methods for preparing monoclonal antibodies.

"Naked antibody" refers to an antibody that is not bound to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. Naked antibodies may be present in pharmaceutical preparations.

"Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, Ig natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as a variant heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variant light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of their constant domains, the light chains of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ).

As used herein, the terms "first," "second," or "third," etc., with respect to an antigen-binding moiety or domain, are used to facilitate the differentiation of each type of moiety or domain when more than one moiety or domain is present. Unless expressly stated, use of these terms is not intended to confer a specific order or direction.

The terms "multispecific" and "bispecific" mean that an antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants. Typically, bispecific antigen-binding molecules contain two antigen-binding sites, each of which is specific for a different epitope. In certain embodiments, a bispecific antigen-binding molecule is capable of binding two epitopes simultaneously, particularly two epitopes expressed on two different cells.

The terms "valency" or "valency" as used herein refer to the presence of a specified number of antigen-binding sites in an antigen-binding molecule. Therefore, the term "monovalent binding to an antigen" means the presence of one (and no more than one) antigen-binding site specific for the antigen in the antigen-binding molecule.

"Antigen binding site" refers to the site of an antigen-binding molecule that provides interaction with an antigen, ie, one or more amino acid residues. For example, the antigen-binding site of an antibody contains amino acid residues from complementarity-determining regions (CDRs). Native immunoglobulin molecules usually have two antigen-binding sites, and Fab molecules usually have a single antigen-binding site.

As used herein, "activating T cell antigen" refers to an antigenic determinant expressed by T lymphocytes (specifically, cytotoxic T lymphocytes) that is capable of inducing or enhancing T cell activation upon interaction with an antigen-binding molecule. Specifically, the interaction of antigen-binding molecules with activating T cell antigens can induce T cell activation by triggering the signaling cascade of the T cell receptor complex. An exemplary activating T cell antigen is CD3. In a specific embodiment, the activating T cell antigen is CD3, specifically the epsilon subunit of CD3 (see UniProt No. P07766 (ed. 130), NCBI RefSeq No. NP_000724.1, which is for human sequences; or UniProt No. Q95LI5 (version 49), NCBI GenBank number BAB71849.1, this is for the macaque [cynomolgus macaque] sequence).

As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes (specifically, cytotoxic T lymphocytes) selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecules Release, cytotoxic activity and expression of activation markers. T cell activating therapeutic agents used in the present invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art as described herein.

"Target cell antigen" as used herein refers to an antigenic epitope present on the surface of a target cell (eg, a cell in a tumor, such as a cancer cell or a cell of the tumor stroma). In a specific embodiment, the target cell antigen is CD20, specifically human CD20 (see UniProt number P11836).

As used herein, "B cell antigen" refers to an antigenic determinant present on the surface of B lymphocytes, specifically malignant B lymphocytes (in which case the antigen is also referred to as "malignant B cell antigen").

As used herein, "T cell antigen" refers to an antigenic determinant present on the surface of T lymphocytes, specifically cytotoxic T lymphocytes.

“Fab molecule” refers to a protein consisting of the VH and CH1 domains of a heavy chain (“Fab heavy chain”) and the VL and CL domains of an immunoglobulin light chain (“Fab light chain”).

"Fusion" means that the components (e.g., Fab molecule and Fc domain subunit) are linked via a peptide bond, either directly or via one or more peptide linkers.

An "effective amount" of an agent is the amount required to cause physiological changes in the cells or tissues to which it is administered.

The "therapeutically effective amount" of a pharmaceutical agent, such as a pharmaceutical composition, refers to an amount that is effective in achieving the desired therapeutic or preventive effect within the required dosage and time period. A therapeutically effective amount of an agent eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease, for example.

"Therapeutic agent" refers to an active ingredient, such as a pharmaceutical composition, which is administered to an individual in an attempt to alter the natural course of a disease in the individual being treated, and which may be used prophylactically or during the course of clinical pathology. "Immunotherapeutic agent" refers to a therapeutic agent that is administered to an individual in an attempt to restore or enhance the individual's immune response, such as an immune response to a tumor.

The term "pharmaceutical composition" refers to a preparation that is in a form effective to permit the biological activity of the active ingredients contained therein and that does not contain additional components that would have unacceptable toxicity to the subject to whom the composition is to be administered.

"Pharmaceutically acceptable carrier" refers to the ingredients in a pharmaceutical composition other than the active ingredients that are not toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "package insert" or "instructions for use" is used to mean the instructions usually included in the commercial packaging of therapeutic products containing the indications, usage, dosage, administration, Information on combination therapies, contraindications and/or warnings.

As used herein, the term "combination therapy" encompasses combined administration (in which two or more therapeutic agents are included in the same or separate formulations); and separate administration, in which case the administration is as defined herein. Reported antibodies may occur before, simultaneously with and/or after administration of the other therapeutic agent(s), preferably one or more antibodies.

"Exchanged" Fab molecule (also known as "Crossfab") means a Fab molecule in which the variable or constant domains of the Fab heavy chain and the Fab light chain are exchanged (i.e., replaced with each other), that is, an exchanged Fab molecule Contains a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1, in the N-terminal to C-terminal direction), and a peptide composed of the heavy chain variable domain VH and the light chain constant domain CL chain (VH-CL, in the N-terminal to C-terminal direction). For the sake of clarity, in exchanged Fab molecules in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (exchanged) Fab molecule. Chain". In contrast, in an exchanged Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (exchanged) Fab molecule.

In contrast, a "conventional" Fab molecule is meant in its natural form (i.e., consisting of a heavy chain (VH-CH1, in the N-to-C-terminal direction) consisting of a heavy chain variable domain and a constant domain) and a light chain variable domain. Fab molecule composed of a light chain (VL-CL, in the N-terminal to C-terminal direction) and a constant domain.

The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), RNA of viral origin, or plastid DNA (pDNA). Polynucleotides may contain conventional phosphodiester linkages or non-conventional linkages (eg, amide bonds, such as found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid fragments, such as DNA or RNA fragments, present in a polynucleotide.

"Isolated nucleic acid molecule or polynucleotide" refers to a nucleic acid molecule (DNA or RNA) that has been separated from its natural environment. For example, for the purposes of this invention, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, as well as plus- and minus-stranded forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include synthetically produced such molecules. Additionally, a polynucleotide or nucleic acid may be or may include regulatory elements, such as a promoter, a ribosome binding site, or a transcription terminator.

By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence. , the polynucleotide sequence may contain up to five point mutations in addition to every 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide with a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or A maximum of 5% of the total number of nucleotides in the reference sequence was inserted into the reference sequence. These changes to the reference sequence may occur at the 5' or 3' end positions of the reference nucleotide sequence or anywhere between these end positions, both interspersed between residues of the reference sequence and within the reference sequence. in one or more consecutive groups. Indeed, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the invention can be determined using This is routinely determined using known computer programs, such as the program discussed above for polypeptides (eg, ALIGN-2).

The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide that has a series of specific nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. Recombinant expression cassettes can be introduced into plastids, chromosomes, mitochondrial DNA, chromosomal DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, expression cassettes of the invention comprise polynucleotide sequences encoding bispecific antigen-binding molecules of the invention, or fragments thereof.

The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene and direct the expression of that gene, to which the DNA molecule is operably linked in a target cell. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into a genome that has been introduced into the host cell. The expression vector of the present invention includes a performance box. Expression vectors transcribe large amounts of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention or a fragment thereof.

As used herein, the term "about" refers to the usual error range for each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself.

"B-cell proliferative disorder" means a disease in which the number of B cells in a patient is greater than the number of B cells in healthy individuals, and specifically in which an increase in the number of B cells is a cause or a marker of the disease. "CD20-positive B-cell proliferative disorders" are B-cell proliferative disorders in which B cells, specifically malignant B cells (other than normal B cells), express CD20.

Exemplary B-cell proliferative disorders include non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL; e.g., relapsed or refractory DLBCL not otherwise specified (NOS), high-grade B cell lymphoma (HGBCL; such as HGBCL NOS, double-hit HGBCL, and triple-hit HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL due to FL (transformed FL; trFL)); follicular lymphoma (FL), including FL grades 1-3b; mantle cell lymphoma (MCL); marginal zone lymphoma (MZL), including spleen, lymph node, or extranodal MZL. In one embodiment, the CD20-positive B cell proliferative disorder is relapsed or refractory NHL (eg, relapsed or refractory DLBCL, relapsed or refractory FL, or relapsed or refractory MCL).

"Refractory disease" is defined as incomplete response to first-line therapy. In one embodiment, refractory disease is defined as failure to respond to prior therapy or relapse within 6 months of such therapy. In one embodiment, refractory disease is characterized by one or more of the following: progressive disease (PD) as best response to first-line therapy, stable disease (SD) as first-line therapy after at least 4 cycles of Therapy (eg, 4 cycles of rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone, also abbreviated R-CHOP) Best response after at least 6 cycles, or partial response (PR) as best response after at least 6 cycles, and disease progression after biopsy-confirmed residual disease or PR. "Recurrent disease" is defined as complete response to first-line therapy. In one embodiment, disease recurrence is confirmed by biopsy. In one embodiment, the patient relapses or becomes unresponsive after at least two prior systemic treatment regimens, including at least one prior anthracycline-containing regimen and at least one anti-CD20-directed therapy.

A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). mouse). Preferably, the subject or individual is a human. In one case, each individual in a group of individuals is a human being. In one case, each individual in the individual reference group is a human being.

"Treatment" as used herein (and its grammatical variants such as "treatment course" or "in treatment") refers to a clinical intervention that attempts to alter the natural course of a disease in a treated individual and may be preventive or clinically Performed during pathological procedures. Desired therapeutic effects include but are not limited to preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the methods of the present invention are used to delay the development of a disease or slow the progression of a disease.

As used herein, "delayed progression" of a disease or disorder means delaying, hindering, slowing, delaying, stabilizing and/or postponing the development of a disease or disorder (e.g., a CD20-positive B-cell proliferative disorder, such as NHL, such as DLBCL). This delay may be of varying lengths depending on the disease being treated and/or the subject's medical history. As will be apparent to those skilled in the art, sufficient or significant delay may actually encompass prevention such that the subject does not develop the disease. For example, in advanced cancers, the development of central nervous system (CNS) metastases may be delayed.

"Reducing" or "inhibiting" means the ability to cause an overall reduction of, for example, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more . For the sake of clarity, the term also includes reduction to zero (or below the detection limit of the analytical method), that is, complete abolition or elimination. In certain embodiments, reduction or inhibition may refer to invariance of scheduled dosing relative to a target dose using a bispecific antibody utilizing an anti-CD20/anti-CD3 bispecific antibody using an ascending dosing regimen of the present invention. Undesirable events such as cytokine-driven toxicity (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), neurotoxicity, and severe tumor lysis syndrome can be reduced or inhibited after treatment. (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and/or central nervous system (CNS) toxicity. In other embodiments, reduction or inhibition may refer to antibody utility functions mediated by the Fc region of the antibody, such utility functions specifically include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody dependent cellular phagocytosis (ADCP). In other embodiments, reduction or inhibition may refer to the CD20-positive B-cell proliferative disorder being treated (e.g., NHL (e.g., DLBCL), FL (e.g., relapsed and/or refractory FL or transformed FL), MCL, high-malignancy symptoms of B-cell lymphoma or PMLBCL), the presence or size of metastases, or the size of the primary tumor.

As used herein, "administering" means a method of administering a dose of a pharmaceutical composition of an anti-CD20/anti-CD3 bispecific antibody to an individual. The pharmaceutical compositions described herein can be administered intravenously (e.g., by intravenous infusion).

As used herein, "buffer" refers to a buffer solution that resists changes in pH through the action of an acid-base conjugate component (also referred to herein as a buffer). In some embodiments, buffers of the invention have a pH in the range of about 5 to about 6. Exemplary buffers for use in the present invention include, but are not limited to, histidine (e.g., histidine HCl), acetate, phosphate, succinate, or combinations thereof. In some embodiments, the histidine acid is histidine hydrochloride (histidine HCl), histidine acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, hydrogen phosphate Dipotassium, tripotassium phosphate or mixtures thereof.

Pharmaceutical compositions according to the invention may also contain one or more tonicity agents. The term "tonicity agent" refers to a pharmaceutically acceptable excipient used to adjust the tonicity of a formulation. The formulation may be hypotonic, isotonic or hypertonic. Isotonicity is generally related to the osmolarity of a solution, typically that of human serum (approximately 250 to 350 mOsmol/kg). The formulations according to the invention may be hypotonic, isotonic or hypertonic, but are preferably isotonic. An isotonic formulation is a liquid or a liquid reconstituted from a solid form, such as from a lyophilized form, and means a solution that has the same tonicity as some other solution to which it is compared, such as physiological saline solution and serum. Suitable tonicity agents include, but are not limited to, salts such as sodium or potassium chloride, glycerol and any component selected from amino acids or sugars, in particular glucose. Tonicity agents are typically used in amounts of approximately ≥200 mM.

Among stabilizers and tonicity agents, there is a group of compounds that act in two ways, i.e. they can be both stabilizers and tonicity agents at the same time. Examples thereof can be found in the group of sugars, amino acids, polyols, cyclodextrins, polyethylene glycols and salts. An example of a sugar that can be both a stabilizer and a tonicity agent is trehalose.

As used herein, "surfactant" refers to a surfactant, preferably a non-ionic surfactant. Examples of surfactants herein include polysorbates (eg, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85) ;Poloxamer (e.g. Poloxamer 188); TRITON®; Sodium octylglycoside; Lauryl-sulfobetaine, Myristyl-sulfobetaine, Linoleyl-sulfobetaine or stearyl-sulfobetaine; lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine or stearyl-sarcosine; linoleyl-betaine, myristine Betaine or Cetyl-Betaine; Lauramidopropyl-Betaine, Cocamidopropyl-Betaine, Linoleamidopropyl-Betaine, Myristamidopropyl-Betaine Base-Betaine, Palmitoyl-Betaine or Isostearyl-Betaine (e.g. Laureamidopropyl-Betaine); Myristidopropyl-Dimethylamine, Palmitoyl-Betaine Aminopropyl-dimethylamine or isostearyl-aminopropyl-dimethylamine; sodium methylcocoyl-taurate or disodium methylcocoyl-taurate; and MONAQUAT™ series (Mona Industries, Paterson, N.J.); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (such as PLURONIC® type block copolymers, such as PLURONIC® F-68); and the like. In one embodiment, the surfactant herein is polysorbate 20 (PS20). In yet another embodiment, the surfactant herein is poloxamer 188 (P188).

A "preservative" is a compound which may optionally be included in a formulation to substantially reduce bacterial action therein, thereby facilitating, for example, the production of a multi-purpose formulation. Examples of potential preservatives include octadecyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride (a mixture of alkyl benzyl ammonium chlorides where the alkyl group is a long chain compound) and benzethonium chloride. Other types of preservatives include aromatic alcohols, such as phenol, butanol, and benzyl alcohol, alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; Cyclohexanol; 3-pentanol and m-cresol. In one embodiment, the preservative herein is benzyl alcohol. In some embodiments, the formulation contains no preservatives.

A "stable" pharmaceutical composition is one in which the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) substantially maintains its physical and/or chemical stability Pharmaceutical formulations, and/or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability as well as its biological activity when stored (eg, frozen storage). The storage period is usually selected based on the expected storage life of the formulation. Various analytical techniques for measuring protein stability are available in the industry and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones , A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured over a selected period of time at a selected amount of light exposure and/or temperature. Stability can be assessed qualitatively and/or quantitatively in a number of different ways, including assessing agglutination formation (e.g., using particle size screening chromatography, by measuring turbidity, and/or by visual inspection); assessing ROS formation (e.g., using particle size screening chromatography, by measuring turbidity, and/or by visual inspection); , by using light stress assay (light stress assay) or 2,2'-azobis(2-formamidinopropane) dihydrochloride (AAPH) stress assay); making anti-CD20/anti-CD3 bispecific antibodies Oxidation of specific amino acid residues (e.g., Met residues of anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab)); by using a cation exchange layer Analysis, imaging capillary isoelectric focusing (icIEF) or capillary zone electrophoresis to evaluate charge heterogeneity; amine-terminal or carboxyl-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact multi-anti-CD20/anti-CD3 bispecifics Antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessment of biological activity or target binding function of anti-CD20/anti-CD3 bispecific antibodies (e.g., T cells and/or B cells); and the like. Instability can involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and/or Trp oxidation), isomerization (e.g., Asp isomerization structuralization), shearing/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, glycosylation differences, and the like.

The term "liquid" as used herein in connection with a formulation according to the present invention means a formulation that is liquid at atmospheric pressure at a temperature of at least about 2 to about 8°C.

In this application, unless otherwise stated, the techniques used can be found in several well-known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press) and PCR Protocols : A Guide to Methods and Applications (Innis et al. 1990. Academic Press, San Diego, CA), and Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Appropriately, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and/or parameters, unless otherwise stated. Therefore, before methods and uses of the present invention are described, it is to be understood that this invention is not limited to the particular methods, protocols, cell lines, animal species or genera, constructs and reagents described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the scope of the appended claims. III.Pharmaceutical compositions

The invention provides pharmaceutical compositions comprising low concentrations of anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) and uses thereof, e.g., for the treatment of B cell proliferation sexually transmitted diseases (eg, non-Hodgkin's lymphoma, NHL). Pharmaceutical compositions of the invention can be formulated to support low concentrations of anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) and to stabilize the antibody during storage and clinical administration Protein loss by adsorption. For low antibody concentrations that require further dilution and processing before clinical administration, adsorption can be an important issue and may result in low potency values. Gerfituzumab was administered at doses of 2.5 mg and 10 mg (stepped dose) and a maintenance dose of 30 mg (target dose, fixed dose). Gerfituzumab is intended for IV administration via IV bag infusion upon dilution in 0.9% or 0.45% sodium chloride. With dosages of 0.05 mg/ml to 0.6 mg/ml solution concentration in IV bag depends on dose.

In one embodiment, a liquid pharmaceutical composition is provided, comprising: About 1 to 25 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab); About 10 to 50 mM buffer; About ≥200 mM tonicity agent; approximately 0 to 15 mM methionine; and Approximately ≥ 0.2 mg/ml surfactant; The pH ranges from about 5.0 to about 6.0.

In one embodiment, a liquid pharmaceutical composition is provided, comprising: Approximately 5 mg/ml of anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab); About 10 to 50 mM buffer; About ≥200 mM tonicity agent; approximately 0 to 15 mM methionine; and Approximately ≥ 0.2 mg/ml surfactant; The pH ranges from about 5.0 to about 6.0.

In one embodiment, a liquid pharmaceutical composition is provided, comprising: About 0.9 to 1.1 mg/ml of anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab); About 10 to 50 mM buffer; About ≥200 mM tonicity agent; approximately 0 to 15 mM methionine; and Approximately ≥ 0.2 mg/ml surfactant; The pH ranges from about 5.0 to about 6.0.

In one embodiment, a liquid pharmaceutical composition is provided, comprising: Approximately 1 mg/ml of anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab); About 10 to 50 mM buffer; About ≥200 mM tonicity agent; approximately 0 to 15 mM methionine; and Approximately ≥ 0.2 mg/ml surfactant; The pH ranges from about 5.0 to about 6.0.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody concentration is in the range of about 1 to 5 mg/ml. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody concentration is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 mg/ml, about 1.1 mg/ml, about 1.5 mg/ml, About 2 mg/ml, about 3 mg/ml, about 4 mg/ml, or about 5 mg/ml. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody concentration is about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg /ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml , about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml, about 26 mg/ml, about 27 mg/ml, about 28 mg/ml, about 29 mg/ml, or about 30 mg/ml.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody concentration is in the range of about 0.9 to 1.1 mg/ml. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody concentration is about 1 mg/ml.

In one embodiment, the liquid pharmaceutical composition comprises an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprising at least one antigen that specifically binds to CD20 A binding domain comprising a heavy chain variable region comprising (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region, which includes (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) of the liquid pharmaceutical composition comprises at least one antigen-binding domain that specifically binds to CD20 , which includes a heavy chain variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence and SEQ ID NO: 7 are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, and the light chain variable region sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 8. In further embodiments, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD20, comprising SEQ. The heavy chain variable region sequence of ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) of the liquid pharmaceutical composition comprises at least one antigen-binding domain that specifically binds to CD3 , which contains: Heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and a light chain variable region, which includes (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) of the liquid pharmaceutical composition comprises at least one antigen-binding domain that specifically binds to CD3 , which includes a heavy chain variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence and SEQ ID NO: 15 are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, and the light chain variable region sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16. In further embodiments, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD3, comprising SEQ. The heavy chain variable region sequence of ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) of the liquid pharmaceutical composition comprises a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) of the liquid pharmaceutical composition comprises (i) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and (ii) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In one embodiment, the antigen-binding domain that specifically binds to CD3 of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is an antibody fragment, specifically Fab molecule or scFv molecule, more specifically Fab molecule. In specific embodiments, the antigen-binding domain that specifically binds to CD3 of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is an exchangeable Fab molecule, wherein The variable or constant domains of the Fab heavy chain and the Fab light chain are exchanged (ie, replaced with each other).

In one embodiment, the antigen-binding domain that specifically binds to CD20 of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is an antibody fragment, specifically Fab molecule or scFv molecule, more specifically Fab molecule. In certain embodiments, the antigen-binding domain that specifically binds to CD20 of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is a conventional Fab molecule.

In one embodiment, the liquid pharmaceutical composition of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD20 and an antigen-binding domain that specifically binds to CD3. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody of the liquid pharmaceutical composition includes a first antigen-binding domain that specifically binds to CD3 and second and third antigen-binding domains that specifically bind to CD20. In one embodiment, the first antigen binding domain is an exchangeable Fab molecule, and the second and third antigen binding domains are each a conventional Fab molecule. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) further comprises an Fc domain. Liquid pharmaceutical compositions of anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) may include modifications in the Fc region and/or antigen-binding domain as described herein . In one embodiment, the liquid pharmaceutical composition of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) includes an IgG1 Fc domain that includes an Fc receptor that reduces One or more amino acid substitutions for binding and/or effector functions. In one embodiment, the liquid pharmaceutical composition of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) includes an IgG1 Fc domain that includes an amino acid substitution L234A, L235A and P329G (EU number).

In one embodiment, the liquid pharmaceutical composition of the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises (i) The antigen-binding domain that specifically binds to CD3 is fused to the N-terminus of the first unit of the Fc domain at the C-terminus of the Fab heavy chain; (ii) a first antigen-binding domain that specifically binds to CD20 fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc heavy chain of an antigen-binding domain that specifically binds to CD3; and (iii) A second antigen-binding domain that specifically binds to CD20, which is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second unit of the Fc domain.

In certain embodiments, the liquid pharmaceutical composition of the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises a) a first Fab molecule that specifically binds to CD3, in particular CD3 epsilon; and in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain replace each other; b) The second Fab and the third Fab molecule, which specifically bind to CD20, wherein in the constant domain CL of the second Fab and the third Fab molecule, the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced (according to Kabat numbering) by lysine (K) or arginine (R), specifically arginine (R), and where in In the constant domain CH1 of the second and third Fab molecules, the amino acid at position 147 is replaced by glutamic acid (E) (EU number), and the amino acid at position 213 is replaced by glutamic acid (E ) replaces (EU number); and c) Fc domain, which consists of the first unit and the second unit that can stably associate.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody of the liquid pharmaceutical composition (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) contains two antigen-binding domains that specifically bind to CD20 and an antigen-binding domain that specifically binds to CD3.

In one embodiment, the liquid pharmaceutical composition of the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is bivalent for CD20 and monovalent for CD3 .

In one embodiment, the first Fab molecule under a) is fused to the N-terminus of one of the subunits in the Fc domain under c) at the C-terminus of the Fab heavy chain, and the second Fab molecule under b) is at the C-terminus of the Fab heavy chain The C-terminus of the heavy chain of the first Fab molecule under a) is fused to the N-terminus of the heavy chain of the first Fab molecule under b) and the C-terminus of the Fab heavy chain under b) is fused to the N-terminus of another subunit of the Fc domain under c) end. In one embodiment, the first Fab molecule under a) comprises: a heavy chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 15; and light A chain variable region that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16.

In another embodiment, the first Fab molecule under a) comprises the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In one embodiment, the second Fab molecule and the third Fab molecule under b) each comprise: a heavy chain variable region that is at least 95%, 96%, 97%, 98% or more identical to the sequence of SEQ ID NO: 7 99% identical; and a light chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 8.

In one embodiment, the second Fab molecule and the third Fab molecule under b) each comprise the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

In a specific embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) of the liquid pharmaceutical composition comprises: a sequence corresponding to at least SEQ ID NO: 17 A polypeptide that is 95%, 96%, 97%, 98% or 99% identical; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 18; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18 : 19 is a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical; and is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 20 of peptides. In another specific embodiment, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO:17, the polypeptide sequence of SEQ ID NO:18, the polypeptide sequence of SEQ ID NO:19 and the polypeptide sequence of SEQ ID NO:20. In another specific embodiment, the bispecific antibody comprises: a polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17; a polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18 sequence; one polypeptide chain, the polypeptide chain comprising the amino acid sequence of SEQ ID NO: 19; and two polypeptide chains, each of the polypeptide chains comprising the amino acid sequence of SEQ ID NO: 20.

Specific anti-CD20/anti-CD3 bispecific antibodies are described in PCT Publication No. WO 2016/020309 and European Patent Application Nos. EP15188093 and EP16169160 (each of which is incorporated herein by reference in its entirety).

In one embodiment, the liquid pharmaceutical composition is an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) that specifically binds to CD3ɛ.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody of the liquid pharmaceutical composition can be combined with antibody H2C (PCT Publication No. WO2008/119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl. 7, 45-50 (1992) and U.S. Patent No. 6,054,297), antibody FN18 (Nooij et al., Eur J Immunol . 19, 981-984 (1986)), antibody SP34 (Pessano et al., EMBO J. 4, 337-340 (1985) ), antibody OKT3 (Kung et al., Science 206, 347-349 (1979)), antibody WT31 (Spits et al., J Immunol . 135, 1922 (1985)), antibody UCHT1 (Burns et al., J Immunol. 129, 1451-1457 (1982)), antibody 7D6 (Coulie et al., EurJ Immunol . 21, 1703-1709 (1991)) or antibody Leu-4. In some embodiments, the anti-CD20/anti-CD3 bispecific antibody of the liquid pharmaceutical composition may also include an antigen-binding moiety that specifically binds to CD3 as disclosed in: WO 2005/040220, WO 2005/118635, WO 2007/042261, WO 2008/119567, WO 2008/119565, WO 2012/162067, WO 2013/158856, WO 2013/188693, WO 2013/186613, WO 2014/110601, WO 2014/145806 ,WO 2014/191113,WO 2014/047231, WO 2015/095392, WO 2015/181098, WO 2015/001085, WO 2015/104346, WO 2015/172800, WO 2016/020444 or WO 2016/014974.

In some embodiments, the anti-CD20/anti-CD3 bispecific antibody of the liquid pharmaceutical composition may include rituximab, obinutuzumab, ocrelizumab, ofatumumab, orocalumab. Antibody or antigen-binding portions of tocilizumab, veltuzumab, and utuximab.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody is gaffetuzumab.

In some embodiments, anti-CD20/anti-CD3 bispecific antibodies may comprise generic, biosimilar, or non-comparable biologic antibody formats as named herein.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody of the liquid pharmaceutical composition provided herein is gaffetuzumab. Gerfituzumab (WHO Drug Information (International Generic Name), Proposed INN: List 83, 2020, Volume 34, Issue 1, Page 39, also known as CD20-TCB, RO7082859 or RG6026; CAS # : 2229047-91-8) is a new type of T cell engaging bispecific (TCB) full-length antibody with a 2:1 molecular configuration that can bivalently bind to CD20 on B cells and bind to CD3 on T cells. (specifically the CD3 epsilon chain (CD3e)) for monovalent binding. Its CD3 binding domain is fused to one of the CD20 binding domains in a head-to-tail manner via a flexible linker. This structure confers superior in vitro potency to other CD20-CD3 bispecific antibodies with a 1:1 configuration and results in significant anti-tumor efficacy in preclinical DLBCL models. CD20 bivalent retains this potency in the presence of competing anti-CD20 antibodies, providing opportunities for pretreatment or co-treatment with these agents. Gefituzumab contains an engineered heterodimeric Fc region that completely eliminates binding to FcgR and C1q. By simultaneously binding to CD3e on tumor cells expressing human CD20 and the T cell receptor (TCR) complex on T cells, in addition to T cell activation, proliferation and cytokine release, it also induces tumor cell lysis. B cell lysis mediated by gaffetuzumab is CD20 specific and does not occur in the absence of CD20 expression or in the absence of simultaneous binding (cross-linking) of T cells to CD20 expressing cells. In addition to killing, T cells are also activated due to CD3 cross-linking, such as through the increase of T cell activation markers (CD25 and CD69), the release of cytokines (IFNγ, TNFα, IL-2, IL-6, IL- 10), cytotoxic granule release (granzyme B) and T cell proliferation were detected. The schematic diagram of the molecular structure of gaffetuzumab is shown in Figure 2. The sequences of gaffetuzumab are summarized in Table 2. Table 2. Sequence ID of gaffetuzumab Sequence ID of gaffetuzumab CD3 heavy chain CD3 light chain SEQ ID NO: instruction SEQ ID NO: instruction 9 HVR-H1 (Kabat) 12 HVR-L1 (Kabat) 10 HVR-H2 (Kabat) 13 HVR-L2 (Kabat) 11 HVR-H3 (Kabat) 14 HVR-L3 (Kabat) 15 VH 16 VL CD20 heavy chain CD20 light chain 1 HVR-H1 (Kabat) 4 HVR-L1 (Kabat) 2 HVR-H2 (Kabat) 5 HVR-L2 (Kabat) 3 HVR-H3 (Kabat) 6 HVR-L3 (Kabat) 7 VH 8 VH full length antibody 17 HC-pestle 18 HC-mortar 19 LC-CD3 20 LC-CD20

In some embodiments, the buffering agent is histidine, acetate, phosphate, succinate, citrate, or combinations thereof. In some embodiments, the histidine acid is histidine acetate. Alternative buffering agents include sodium phosphate monobasic, disodium hydrogen phosphate, trisodium phosphate, potassium monobasic phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, or mixtures thereof. In certain embodiments, the liquid pharmaceutical composition comprises a histidine buffer, ie, a buffer having histidine, typically L-histidine, as a buffering agent. In a specific embodiment, the buffer comprises L-histidine HCl, ie a buffer comprising L-histidine or a mixture of L-histidine and L-histidine HCl, and the pH adjustment is achieved with hydrochloric acid. L-Histidine HCl buffer can be prepared by dissolving an appropriate amount of L-histidine acid and L-histamine hydrochloride in water, or by dissolving an appropriate amount of L-histamine acid in water and adjusting it by adding hydrochloric acid. pH to the desired value.

In some examples, the concentration of the buffer (e.g., histidine, e.g., L-histidine HCl) is 10 mM to 50 mM. For example, the buffer can be 10 mM to 15 mM, or 15 mM to 20 mM, such as 6 mM to 18 mM, 7 mM to 16 mM, 8 mM to 15 mM, or 9 mM to 12 mM, such as about 10 mM , about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM. In specific examples, the concentration of the buffer (e.g., histidine, e.g., L-histidine HCl) is about 15 to 25 mM. In one embodiment, the concentration of the buffer (e.g., histidine, e.g., L-histidine HCl) is about 20 mM.

Regardless of the buffer used, the pH can be adjusted to a value in the range of about 5.0 to about 6.0 using acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide. , specifically adjusted to a pH of about 5.2 to about 5.8.

The inventors of the present invention discovered that anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) contain a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14, Particularly stable in compositions at a pH of about 5.2 to about 5.8. In one embodiment, the buffer provides a pH of about 5.2 to about 5.8, specifically a pH of about 5.5.

In some embodiments, pharmaceutical compositions include a tonicity agent, such as a sugar, amino acid, or salt. In embodiments where the tonicity agent is a sugar, the sugar may be, for example, sucrose, glucose, glycerol or trehalose. In specific embodiments, the sugar is sucrose, optionally D-sucrose. In some embodiments, the tonicity agent is sucrose or sodium chloride. The concentration of the tonicity agent (e.g., sugar, e.g., sucrose) can be at least about ≥200 mM. For example, the tonicity agent (e.g., sugar, such as sucrose) can be at, e.g., 200 to 220 mM, 220 to 240 mM, 240 to 260 mM, 260 to 280 mM, 280 to 300 mM, 300 to 320 mM or 480mM to 500mM, for example, 200mM to 300mM, for example, about 200mM, about 210mM, about 220mM, about 230mM, about 240mM, about 250mM, about 260mM, about 270mM, about 280 a concentration of about 290 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM or about 500 mM. In some embodiments, the concentration of tonicity agent is about 200 mM to 280 mM. In some embodiments, the concentration of tonicity agent is about 240 mM. In a specific embodiment, the tonicity agent is sucrose and is present at a concentration of at least about 200 mM, ie, at a concentration of about ≥200 mM. In other specific embodiments, the tonicity agent is sucrose (e.g., D-sucrose) and is present at a concentration of about 200 mM to 280 mM. In a specific embodiment, the tonicity agent is sucrose (e.g., D-sucrose) and is present at a concentration of about 240 mM. In some embodiments, the liquid pharmaceutical composition includes methionine as a stabilizer.

Any suitable concentration of the stabilizer methionine may be used. For example, in some embodiments of any of the aforementioned pharmaceutical compositions, the concentration of the stabilizer (e.g., methionine) is from about 0.01 mM to about 15 mM, for example, about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, About 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM or about 15mM.

In specific embodiments, the concentration of methionine is about 5 mM to 15 mM. In specific embodiments, the concentration of methionine is about 10 mM.

Any pharmaceutical composition described herein may include surfactants. Any suitable surfactant can be used. In some embodiments, the surfactant is a nonionic surfactant (e.g., polysorbate (polyoxyethylene(n) sorbitol monolaurate), poloxamer, polyoxyethylene alkyl ether, Alkylphenyl polyoxyethylene ether or combination thereof). In some embodiments, the nonionic surfactant is a polysorbate (e.g., polysorbate 20 (polyoxyethylene (20) sorbitol monolaurate (PS20)), TWEEN 20®; e.g., ultra-refined PS20 (PS20 that has undergone a proprietary flash chromatography process to achieve higher purity and is available from Avantor Performance Materials, LLC (Center Valley, PA, US)) or Polysorbate 80 (Polyoxyethylene (20) Sorbate Sugar alcohol monooleate (PS80), e.g. TWEEN 80®; e.g. ultra-refined PS80 (Avantor))). In a specific embodiment, the polysorbate is polysorbate 20. In other embodiments, the nonionic surfactant is a poloxamer (e.g., poloxamer 188, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) .

The concentration of the pharmaceutical surfactant may be at least about ≥ 0.2 mg/ml, i.e., at a concentration of at least about ≥ 0.02% (w/v).

In some embodiments of any pharmaceutical composition described herein, the surfactant (e.g., PS20 or P188) is present at a concentration of about 0.01% (w/v) to about 2% (w/v), for example, about 0.01% , about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6% , about 1.7%, about 1.8%, about 1.9% or about 2% (w/v).

In some embodiments, the surfactant (e.g., PS20 or P188) is present at a concentration of about 0.1 to 1 mg/ml, that is, 0.01% (w/v) to about 0.1% (w/v). In some embodiments, the surfactant (e.g., PS20 or P188) is present at a concentration of about 0.2 to 1 mg/ml, that is, 0.02% (w/v) to about 0.1% (w/v). In some embodiments, the concentration of surfactant (e.g., PS20 or P188) is about 0.2 to 0.8 mg/ml, that is, 0.02% (w/v) to about 0.08% (w/v). In some embodiments, the surfactant (e.g., PS20 or P188) is present at a concentration of about 0.5 mg/ml, or 0.05% (w/v).

In certain embodiments, the surfactant is P188, and the concentration of P188 is about 0.05% (w/v), 0.07% (w/v), or 0.1% (w/v).

In a specific embodiment, the surfactant is PS20, and the concentration of PS20 is at least about ≥ 0.2 mg/ml, that is, the concentration is at least about ≥ 0.02% (w/v) PS20.

In a specific embodiment, the surfactant is PS20, and the concentration of PS20 is about 0.2 to 0.8 mg/ml, that is, about 0.02% (w/v) to about 0.08% (w/v). In a specific embodiment, the surfactant is PS20, and the concentration of PS20 is about 0.5 mg/ml, or 0.05% (w/v). In certain embodiments, the surfactant is PS20, and the concentration of PS20 is at least about ≥ 0.02% (w/v) PS20. In a specific embodiment, the surfactant is PS20, and the concentration of PS20 is about 0.02% (w/v) to about 0.08% (w/v). In a specific embodiment, the surfactant is PS20, and the concentration of PS20 is about 0.05% (w/v).

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 to 5 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5 to about 6.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 to 5 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 0.9 to 1.1 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition includes: Approximately 1 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition contains: Approximately 1 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: a) At least one antigen-binding domain that specifically binds to CD20 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3 It contains a heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14; Approximately 20 mM histamine buffer; Approximately 240 mM sucrose; Approximately 10 mM methionine; and Approximately 0.5 mg/ml of PS20 The pH is about 5.5.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 to 5 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: (i) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and (ii) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: About 0.9 to about 1.1 mg/ml of an anti-CD20/anti-CD3 bispecific antibody, comprising: (i) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and (ii) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: (i) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and (ii) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 mg/ml of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) containing: (i) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and (ii) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; Approximately 20 mM histamine buffer; Approximately 240 mM sucrose; Approximately 10 mM methionine; and Approximately 0.5 mg/ml of PS20 The pH is about 5.5.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 to 5 mg/ml of greffitumab, Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: about 0.9 to about 1.1 mg/ml of greffitumab, Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 mg/ml of greffitumab; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5.2 to about 5.8.

In one embodiment, the liquid pharmaceutical composition according to the present invention contains: Approximately 1 mg/ml of greffitumab; Approximately 20 mM histamine buffer; Approximately 240 mM sucrose; Approximately 10 mM methionine; and Approximately 0.5 mg/ml of PS20 The pH is approximately 5.5.

The formulations may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured through sterilization procedures and by adding various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Preservatives are typically used in amounts from about 0.001 to about 2% (w/v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chlorom-cresol, methyl or propylparaben, and benzalkonium chloride. IV. Therapeutic agents used in the pharmaceutical compositions of the present invention A. Anti -CD20/ anti -CD3 bispecific antibodies

The present invention provides new pharmaceutical compositions of anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 T cell engaging bispecific antibodies (TCB), e.g., gaffetuzumab). In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody is a polyclonal antibody. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody is a human antibody. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is a humanized antibody. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody is a chimeric antibody. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody is a full-length antibody. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is an IgG class antibody, specifically an IgGl subclass antibody. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is a recombinant antibody.

In certain embodiments, anti-CD20/anti-CD3 bispecific antibodies comprise antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, as well as others described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see for example Pluckthün, The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and USA Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments that contain salvage receptor binding epitope residues and have increased half-life in vivo, see US Patent No. 5,869,046. In one embodiment, the antibody fragment is a Fab fragment or a scFv fragment.

Bifunctional antibodies are antibody fragments with two antigen-binding sites (which may be bivalent or bispecific). See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1 No.).

Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as disclosed herein and production of recombinant host cells (eg, E. coli or phage).

In certain embodiments, anti-CD20/anti-CD3 bispecific antibodies are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA , 81: 6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed compared to its parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the anti-CD20/anti-CD3 bispecific antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies contain one or more variable domains, where the HVRs such as CDRs (or portions thereof) are derived from the non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies will optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in: Riechmann et al. , Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (described in detail determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling");Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer , 83:252-260 (2000) (describing "guided selection" of FR shuffling Law).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using a "best match" approach (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); Framework regions of consensus sequences for human antibodies of specific subgroups of chain variant regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA , 89:4285 (1992); and Presta et al. J. Immunol. , 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and from screening FR libraries framework region (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

In certain embodiments, the anti-CD20/anti-CD3 bispecific antibody is a human antibody. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in: van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001); and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, either present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example: U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE ^™ technology); U.S. Patent No. 5,770,429 (describing HuMab® technology); U.S. Patent No. 7,041,870 (describing KM MOUSE® technology); and U.S. Patent Application Publication No. US 2007/0061900 (describing VelociMouse® technology). The human variable regions derived from intact antibodies produced by such animals can be further modified, for example, by binding to different human constant regions.

Human antibodies can also be produced through fusionoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines have been described for the production of human monoclonal antibodies. (See, e.g., Kozbor J. Immunol. , 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al. , J. Immunol ., 147: 86 (1991).) Human antibodies generated via human B cell fusionoma technology are also described in Li et al ., Proc. Natl. Acad. Sci. USA , 103:3557-3562 (2006) middle. Other methods include those described in, for example, US Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from fusionoma cell lines), and Ni, Xiandai Mianyixue , 26(4):265-268 (2006) ( Human-human fusion tumors are described). Human fusion tumor technology (Trioma technology) is also described in: Vollmers and Brandlein, Histology and Histopathology , 20(3):927-937 (2005); and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology , 27 (3):185-91 (2005).

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human phage display libraries. These variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

Binding domains comprised in anti-CD20/anti-CD3 bispecific antibodies (eg, anti-CD20/anti-CD3 TCB, eg, gaffetumumab) can be isolated by screening combinatorial libraries of binding moieties with the desired activity. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are reviewed, for example, by Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, by McCafferty et al. Nature 348:552-554; Clackson et al. Nature 352: 624-628 (1991); Marks et al. J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al. J. Mol. Biol. 338(2): 299-310 (2004); Lee et al. J. Mol. Biol. 340(5 ): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1-2): 119- 132 (2004).

In some phage display methods, VH and VL gene libraries are selected separately through polymerase chain reaction (PCR) and randomly recombined in the phage library, and then antigen-binding phages can be screened according to the method described in the following literature: Winter et al. Human, Ann. Rev. Immunol. , 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies to the immunogen without the need to construct fusionomas. Alternatively, natural lineages (e.g., from humans) can be selected without any immunization to provide a single source of antibodies to various non-self as well as self-antigens, as described by Griffiths et al. in EMBO J. 12: 725-734 ( 1993). Finally, natural libraries such as Hoogenboom and Winter can be synthesized by selecting unrearranged V gene fragments in stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and completing the rearrangement in vitro . Described in J. Mol. Biol. , 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2 009 /0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered herein to be human antibodies or human antibody fragments.

Techniques for making bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see: Milstein et al., Nature 305: 537 (1983); WO 93/08829 ; and Traunecker et al., EMBO J. 10: 3655, 1991) and "mortar and pestle" engineering modifications (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimer molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g. , U.S. Patent No. 4,676,980; and Brennan et al ., Science , 229: 81 (1985)); use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. , 148(5) :1547-1553 (1992)); using "diabody" technology to prepare diabody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. , 152:5368 (1994)); and according to, e.g., Tutt et al., J. Immunol. 147:60 (1991) The disclosed methods prepare trispecific antibodies.

Also included herein are engineered antibodies with three or more antigen binding sites, including "Octopus antibodies" (see, eg, US 2006/0025576A1).

Anti-CD20/anti-CD3 bispecific antibodies herein also include "dual-action FAbs" or "DAFs," which contain antigen-binding sites that bind two different antigens (see, e.g., US 2008/0069820).

"Crossmab" antibodies are also included herein (see, e.g., WO2009080251, WO2009080252, WO2009080253, WO2009080254).

Another technology used to prepare bispecific antibody fragments is the "bispecific T cell engagement" or BiTE® approach (see, for example, WO2004/106381, WO2005/061547, WO2007/042261 and WO2008/119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain includes two single-chain Fv (scFv) fragments, each having a variable heavy (VH) domain and a variable light (VL) domain separated by a polypeptide linker that links The length of the subdomain is sufficient to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, so when each scFv engages its cognate epitope, cells of two different cell types are brought into close proximity or Trapped. A specific example of this approach includes an scFv that recognizes a cell surface antigen expressed by an immune cell (e.g., a CD3 polypeptide on a T cell) linked to another scFv that recognizes a cell surface antigen expressed by a target cell (e.g., a malignant or tumor cell) Surface antigen scFv.

Because it is a single polypeptide, any prokaryotic or eukaryotic cell expression system known in the art (such as CHO cell lines) can be used to express the bispecific T cell engager. However, specific purification techniques (see, e.g., EP1691833) may be required to separate monomeric bispecific T cell engagers from other multimeric species that may have biological activities in addition to the intended monomeric activity. In an exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using an imidazole concentration gradient. This eluate was further purified using anion exchange chromatography, and the peptide was eluted using a sodium chloride concentration gradient. Finally, the eluate is subjected to particle size screening chromatography to separate monomeric and polymeric species.

In certain embodiments, anti-CD20/anti-CD3 bispecific antibodies can be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for derivatization of anti-CD20/anti-CD3 bispecific antibodies (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetumumab) include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, Poly-1,3-dioxolane, poly-1,3,6-triethane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylenated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used in the specified conditions. The treatment below is moderate.

Anti-CD20/anti-CD3 bispecific antibodies can also bind to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatic toxins of bacterial, fungal, plant or animal origin) or fragments thereof), or radioactive isotopes.

In one embodiment, anti-CD20/anti-CD3 bispecific antibodies comprise antibody-drug conjugates (ADCs) in which the antibody is conjugated to one or more drugs including, but not limited to, maytansinoid ( See U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0425235 B1); auristatin, such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and Nos. 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, and Nos. 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); Anthracyclines, such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; and CC1065.

In another embodiment, the immunoconjugate comprises an anti-CD20/anti-CD3 bispecific antibody that binds to an enzymatic toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding diphtheria toxin Active fragment, exotoxin A chain (derived from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, α-sarcina, tung protein, aromatic acid Caryophyllin, Phytolactin (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin ), mitogellin, restrictocin, phenomycin, enomycin and crescentin.

In another embodiment, an anti-CD20/anti-CD3 bispecific antibody binds to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes can be used to produce radioactive conjugates. Examples include the radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When radioconjugates are used for detection, they may contain radioactive atoms such as Tc ^99m or I ¹²³ for scintigraphy studies, or natural atoms for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI). Spin labels such as iodine-123 (again), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gallium, manganese or iron.

Conjugates of anti-CD20/anti-CD3 bispecific antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein couplers, such as N-succinimidyl-3-(2-pyridine Sulfonyl dithio)propionate (SPDP), succinimidyl-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate HCl), active esters (e.g., disuccinimidyl suberic acid), aldehydes (e.g., glutaraldehyde), bisazide Compounds (e.g. bis(p-azidobenzyl)hexanediamine), bis-nitrogen derivatives (e.g. bis-(p-diazobenzyl)-ethylenediamine), diisocyanates (e.g. toluene 2,6 -diisocyanate) and dual-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). One exemplary chelating agent used to conjugate radioactive nucleotides to antibodies is carbon-14 labeled benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA). See WO94/11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs from cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52:127 -131 (1992); U.S. Patent No. 5,208,020).

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is suitable for treating a cell proliferative disorder (e.g., cancer). In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cancer is a B cell proliferative disorder. In one embodiment, the cancer is a CD20-positive B cell proliferative disorder. In one embodiment, the cancer is non-Hodgkin's lymphoma (NHL). In one embodiment, NHL is diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), DLBCL derived from follicular lymphoma (FL) [transformed FL; trFL], original idiopathic mediastinal large B-cell lymphoma (PMBCL) or marginal zone lymphoma (MZL). MZL can be classified into splenic MZL, nodular MZL, and extranodular MZL. In one embodiment, NHL is mantle cell lymphoma (MCL). In one embodiment, the NHL is grade 1-3a follicular lymphoma (FL). In one embodiment, the CD20-positive B cell proliferative disorder is a relapsed or refractory B cell proliferative disorder. In one embodiment, the relapsed or refractory B-cell proliferative disorder is relapsed or refractory NHL (eg, relapsed or refractory DLBCL, relapsed or refractory FL, or relapsed or refractory MCL).

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) specifically binds to CD3ɛ.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody can be combined with antibody H2C (PCT Publication No. WO 2008/119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl. 7, 45-50 (1992) and U.S. Patent No. 6,054,297), antibody FN18 (Nooij et al., Eur J Immunol . 19, 981-984 (1986)), antibody SP34 (Pessano et al., EMBO J. 4, 337-340 (1985)), antibody OKT3 (Kung et al., Science 206, 347-349 (1979)), antibody WT31 (Spits et al., J Immunol . 135, 1922 (1985)), antibody UCHT1 (Burns et al., J Immunol. 129, 1451-1457 ( 1982)), antibody 7D6 (Coulie et al., EurJ Immunol . 21, 1703-1709 (1991)) or antibody Leu-4. In some embodiments, anti-CD20/anti-CD3 bispecific antibodies may also comprise an antigen-binding moiety that specifically binds to CD3 as disclosed in: WO 2005/040220, WO 2005/118635, WO 2007/042261, WO 2008/119567、WO 2008/119565、WO 2012/162067、WO 2013/158856、WO 2013/188693、WO 2013/186613、WO 2014/110601、WO 2014/145806、WO 2014/191113 ,WO 2014/047231,WO 2015/095392, WO 2015/181098, WO 2015/001085, WO 2015/104346, WO 2015/172800, WO 2016/020444 or WO 2016/014974.

In some embodiments, the anti-CD20/anti-CD3 bispecific antibody may comprise rituximab, obinutuzumab, ocrelizumab, ofatumumab, ocratuzumab ( ocaratuzumab), veltuzumab (veltuzumab), and ublituximab (ublituximab) antibody or antigen-binding portion.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody is gaffetuzumab.

In some embodiments, anti-CD20/anti-CD3 bispecific antibodies may comprise generic, biosimilar, or non-comparable biologic antibody formats as named herein.

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD20, comprising: Heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6.

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD20, comprising a heavy chain A variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7 Identical, and the light chain variable region sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 8. In further embodiments, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD20, comprising SEQ. The heavy chain variable region sequence of ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD3, the antigen-binding domain Include Heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14.

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD3, comprising a heavy chain A variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence and SEQ ID NO: 15 are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% Identical, and the light chain variable region sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16. In further embodiments, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD3, comprising SEQ. The heavy chain variable region sequence of ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises: a) At least one antigen-binding domain that specifically binds to CD20, which includes a heavy chain variable region that includes: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3, which includes a heavy chain variable region that includes: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises: (i) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and (ii) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In one embodiment, the antigen-binding domain that specifically binds to CD3 of the anti-CD20/anti-CD3 bispecific antibody is an antibody fragment, particularly a Fab molecule or a scFv molecule, more particularly a Fab molecule. In specific embodiments, the antigen-binding domain of an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) that specifically binds to CD3 is an exchangeable Fab molecule, wherein The variable or constant domains of the Fab heavy chain and the Fab light chain are exchanged (ie, replaced with each other).

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises at least one antigen-binding domain that specifically binds to CD20 and one that specifically binds to CD3 Sexually binding antigen-binding domain. In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises a first antigen-binding domain that specifically binds to CD3 and a first antigen-binding domain that specifically binds to CD20 Binding of the second and third antigen binding domains. In one embodiment, the first antigen binding domain is an exchangeable Fab molecule, and the second and third antigen binding domains are each a conventional Fab molecule. In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) further comprises an Fc domain. Anti-CD20/anti-CD3 bispecific antibodies may contain modifications in the Fc region and/or the antigen-binding domain as described herein. In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) includes an IgG1 Fc domain that includes reduced binding to Fc receptors and/ or one or more amino acid substitutions for effector function. In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (EU number).

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises: (i) The antigen-binding domain that specifically binds to CD3 is fused to the N-terminus of the first unit of the Fc domain at the C-terminus of the Fab heavy chain; (ii) a first antigen-binding domain that specifically binds to CD20 fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc heavy chain of an antigen-binding domain that specifically binds to CD3; and (iii) A second antigen-binding domain that specifically binds to CD20, which is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second unit of the Fc domain.

In specific embodiments, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) comprises: a) a first Fab molecule that specifically binds to CD3, in particular CD3 epsilon; and in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain replace each other; b) The second Fab and the third Fab molecule, which specifically bind to CD20, wherein in the constant domain CL of the second Fab and the third Fab molecule, the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced (according to Kabat numbering) by lysine (K) or arginine (R) (especially arginine (R)), and where at In the constant domain CH1 of the second and third Fab molecules, the amino acid at position 147 is replaced by glutamic acid (E) (EU number), and the amino acid at position 213 is replaced by glutamic acid (E ) replaces (EU number); and c) Fc domain, which consists of the first unit and the second unit that can stably associate.

In one embodiment, an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) contains two antigen-binding domains that specifically bind to CD20 and one that specifically binds to CD3 Sexually binding antigen-binding domain.

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) is bivalent for CD20 and monovalent for CD3.

In one embodiment, a first Fab molecule under a) is fused to the N-terminus of one of the subunits of the Fc domain under c) at the C-terminus of the Fab heavy chain, and b) a second Fab molecule under The C-terminus of the chain is fused to a) the N-terminus of the heavy chain of the first Fab molecule below, and, b) the C-terminus of the third Fab molecule below the Fab heavy chain is fused to c) another subsection of the Fc domain N terminal of the unit. In one embodiment, the first Fab molecule under a) comprises a heavy chain variable region and a light chain variable region that are at least 95%, 96%, 97% identical to the sequence of SEQ ID NO: 15 %, 98% or 99% identical, and the light chain variable region is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16.

In another embodiment, the first Fab molecule under a) comprises the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In one embodiment, the second Fab molecule and the third Fab molecule under b) each comprise: a heavy chain variable region that is at least 95%, 96%, 97%, 98% or more identical to the sequence of SEQ ID NO: 7 99% identical; and a light chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 8.

In one embodiment, the second Fab molecule and the third Fab molecule under b) each comprise the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

In a specific embodiment, the anti-CD20/anti-CD3 bispecific antibody comprises: a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 17; : The sequence of SEQ ID NO: 18 is at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide; the sequence of SEQ ID NO: 19 is at least 95%, 96%, 97%, 98% or 99% identical. A polypeptide; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 20. In another specific embodiment, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO:17, the polypeptide sequence of SEQ ID NO:18, the polypeptide sequence of SEQ ID NO:19 and the polypeptide sequence of SEQ ID NO:20. In another specific embodiment, the bispecific antibody comprises: a polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17; a polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18 sequence; one polypeptide chain, the polypeptide chain comprising the amino acid sequence of SEQ ID NO: 19; and two polypeptide chains, each of the polypeptide chains comprising the amino acid sequence of SEQ ID NO: 20.

Specific anti-CD20/anti-CD3 bispecific antibodies are described in PCT Publication No. WO 2016/020309 and European Patent Application Nos. EP15188093 and EP16169160 (each incorporated herein by reference in its entirety). In one embodiment, the anti-CD20/anti-CD3 bispecific antibody of the pharmaceutical composition of the present invention is gaffetuzumab. B. Antibody format 1. Configuration of anti -CD20/ anti- CD3 bispecific antibody

The components of anti-CD20/anti-CD3 bispecific antibodies can be fused to each other in various configurations. An example configuration is shown in Figure 1.

In certain embodiments, the antigen-binding moiety comprised in the anti-CD20/anti-CD3 bispecific antibody is a Fab molecule. In these embodiments, the first antigen-binding portion, the second antigen-binding portion, the third antigen-binding portion, etc. may be referred to herein as a first Fab molecule, a second Fab molecule, a third Fab molecule, etc., respectively. Additionally, in certain embodiments, the anti-CD20/anti-CD3 bispecific antibody includes an Fc domain composed of a first unit and a second unit capable of stable association.

In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit or the second unit of the Fc domain.

In one such embodiment, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In one specific such embodiment, the anti-CD20/anti-CD3 bispecific antibody consists essentially of a first Fab molecule and a second Fab molecule, an Fc domain consisting of a first unit and a second unit, and optionally one or It consists of multiple peptide linkers, in which the first Fab molecule is fused to the N-terminus of the first unit or the second unit of the Fc domain at the C-terminus of the Fab heavy chain, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain. to the N-terminus of the Fab heavy chain of the first Fab molecule. Such a configuration is schematically depicted in Figures 1G and 1K. Additionally, optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In another embodiment, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit or the second unit of the Fc domain. In a specific such embodiment, the antibody consists essentially of a first Fab molecule and a second Fab molecule, an Fc domain consisting of a first unit and a second unit, and optionally one or more peptide linkers, The first Fab molecule and the second Fab molecule are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain. This configuration is schematically illustrated in Figures 1A and 1D. The first Fab molecule and the second Fab molecule can be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the first Fab molecule and the second Fab molecule are each fused to an Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human _IgG1 hinge region, in particular, wherein the Fc domain is an _IgG1 Fc domain.

In other embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit or the second unit of the Fc domain. In one such embodiment, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific such embodiment, the antibody consists essentially of a first Fab molecule and a second Fab molecule, an Fc domain consisting of a first unit and a second unit, and optionally one or more peptide linkers, The first Fab molecule is fused to the N-terminus of the Fab heavy chain at the C-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused to the first unit or the second unit of the Fc domain at the C-terminus of the Fab heavy chain. The N terminal of the subunit. This configuration is schematically illustrated in Figures 1H and 1L. Additionally, optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

Fab molecules can be fused to the Fc domain directly to each other or to the Fc domain via a peptide linker that contains one or more amino acids and typically has about 2-20 amino acids. Peptide linkers are well known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G ₄ S) _n (SEQ ID NO: 21), (SG ₄ ) _n (SEQ ID NO: 22) or G ₄ (SG ₄ ) _n (SEQ ID NO: 23) Peptide linker. "N" is usually an integer from 1 to 10, especially 2 to 4. In one embodiment, the peptide linker is at least 5 amino acids in length; in one embodiment, from 5 to 100 amino acids in length; in a further embodiment, from 10 to 50 amino acids in length. Amino acids. In one embodiment, the peptide linker is (GxS) _n or (GxS) _n G _m , where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3) (SEQ ID NOs: 27-58), In one embodiment, x=4 and n=2 or 3, in a further embodiment, x=4 and n=2. In one embodiment, the peptide linker is (G ₄ S) ₂ (SEQ ID NO: 24). One particularly suitable peptide linker for fusing the Fab light chains of the first Fab molecule and the second Fab molecule to each other is (G ₄ S) ₂ (SEQ ID NO: 24). An exemplary peptide linker suitable for joining the Fab heavy chain of the first Fab fragment and the second Fab fragment includes the sequence (D)-( _G4S ) ₂ (SEQ ID NO: 24 and 25). Another suitable such linker includes the sequence (G ₄ S) ₄ (SEQ ID NO: 26). Additionally, the linker may comprise (part of) the immunoglobulin hinge region. Specifically, in the case where the Fab molecule is fused to the N-terminus of the Fc domain subunit, the fusion may be through the immunoglobulin hinge region or a portion thereof, which may or may not contain an additional peptide linker.

Antibodies with a single antigen-binding moiety (such as a Fab molecule) that are capable of specifically binding to the target cell antigen can be used (eg, as shown in Figures 1A, 1D, 1G, 1H, 1K, or 1L), particularly where high affinity is expected When the target cell antigen is internalized after binding to the sexual antigen-binding moiety. In such cases, the presence of more than one antigen-binding moiety for a particular target cell antigen may enhance the internalization of the target cell antigen, thereby reducing its availability.

However, in many other cases, antibodies containing two or more antigen-binding portions (such as Fab molecules) specific for a particular target cell antigen (e.g., Figures 1B, 1C, 1E, 1F, 1I , 1J, 1M or 1N) would be advantageous, for example to optimize targeting of target sites or to cross-link target cell antigens.

Accordingly, in certain embodiments, an anti-CD20/anti-CD3 bispecific antibody comprises two anti-CD20 binding moieties, e.g., two CD20-targeting Fab molecules. In one embodiment, the two CD20-targeting Fab molecules are conventional Fab molecules. In one embodiment, two CD20-targeting Fab molecules contain the same heavy and light chain amino acid sequences and have the same domain arrangement (i.e., conventional or swapped).

In alternative embodiments, an anti-CD20/anti-CD3 bispecific antibody comprises two anti-CD3 binding moieties, e.g., two CD3-targeting Fab molecules. In one such embodiment, both CD3-targeting Fab molecules are crossover Fab molecules (a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged with each other/ replacement). In one such embodiment, two CD3-targeting Fab molecules contain the same heavy and light chain amino acid sequences and have the same domain arrangement (i.e., conventional or swapped).

In one embodiment, a third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit or the second unit of the Fc domain.

In a specific embodiment, the second Fab molecule and the third Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first Fab molecule is at the C-terminus of the Fab heavy chain Fusion with the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific such embodiment, the antibody consists essentially of a first Fab molecule, a second Fab molecule, and a third Fab molecule, an Fc domain consisting of a first unit and a second unit, and optionally one or more Composed of a peptide linker, in which the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the first portion of the Fc domain The N-terminus of the second unit of the Fc domain, and the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Such configurations are schematically illustrated in Figures 1B and 1E (Examples in which the third Fab molecule is a conventional Fab molecule and identical to the second Fab molecule) and Figures 1I and 1M (Examples in which the third Fab molecule is an exchange type Fab molecule and preferably the same as the first Fab molecule). The second Fab molecule and the third Fab molecule can be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the second Fab molecule and the third Fab molecule are each fused to the Fc domain through the immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human _IgG1 hinge region, in particular, wherein the Fc domain is an _IgG1 Fc domain. Additionally, optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In another embodiment, the second Fab molecule and the third Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first Fab molecule is at the C-terminus of the Fab heavy chain end is fused to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific such embodiment, the antibody consists essentially of a first Fab molecule, a second Fab molecule, and a third Fab molecule, an Fc domain consisting of a first unit and a second unit, and optionally one or more Composed of a peptide linker, in which the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the first portion of the Fc domain The N-terminus of the second unit of the Fc domain, and the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Such configurations are schematically illustrated in Figures 1C and 1F (Examples in which the third Fab molecule is a conventional Fab molecule and identical to the second Fab molecule) and Figures 1J and 1N (Examples in which the third Fab molecule is an exchange type Fab molecule and is the same as the first Fab molecule) depicted in. The first Fab molecule and the third Fab molecule can be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the second Fab molecule and the third Fab molecule are each fused to the Fc domain through the immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human _IgG1 hinge region, in particular, wherein the Fc domain is an _IgG1 Fc domain. Additionally, optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In an antibody configuration in which a Fab molecule is fused at the C-terminus of the Fab heavy chain via the immunoglobulin hinge region to the N-terminus of each of the Fc domain subunits, two Fab molecules, the hinge region, and the Fc domain essentially form Immunoglobulin molecules. In certain embodiments, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more specific embodiment, the immunoglobulin is an IgG subclass ₁ immunoglobulin. In another embodiment, the immunoglobulin is an IgG subclass ₄ immunoglobulin. In another specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.

In some antibodies, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. According to the different configurations of the first Fab molecule and the second Fab molecule, the Fab light chain of the first Fab molecule can be fused at its C-terminus with the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule can be fused to the N-terminus of the Fab light chain of the second Fab molecule. The chain can be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusion of the Fab light chain of the first Fab molecule to the second Fab molecule further reduces mismatching of mismatched Fab heavy and light chains and also reduces the number of plasmids required to express some antibodies.

In certain embodiments, the antibody comprises a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises exchanged Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VL ₍₁₎ -CH1 ₍₁₎ -CH2-CH3(- CH4)); and a polypeptide, wherein the Fab heavy chain of the second Fab molecule and the Fc domain subunit share a carboxyl terminal peptide bond (VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule and the Fab light chain constant region of the first Fab molecule share a carboxy-terminal peptide bond (VH ₍₁₎ -CL ₍₁ ) ₎ ), and shares a carboxyl-terminal peptide bond (VL ₍₂₎ -CL ₍₂₎ ) with the Fab light chain polypeptide of the second Fab molecule. In certain embodiments, the polypeptides are covalently linked, for example, via disulfide bonds.

In certain embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises exchangeable Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH ₍₁₎ -CL ₍₁₎ -CH2-CH3(-CH4) ); and a polypeptide, wherein the Fab heavy chain of the second Fab molecule shares a carboxyl-terminal peptide bond (VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)) with the Fc domain subunit. In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the first Fab molecule and the Fab heavy chain constant region of the first Fab molecule share a carboxyl-terminal peptide bond (VL ₍₁₎ -CH1 _{(1 )} ), and shares the carboxyl terminal peptide bond (VL ₍₂₎ -CL ₍₂₎ ) with the Fab light chain polypeptide of the second Fab molecule. In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

In some embodiments, the antibody comprises a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises an exchange-type Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit ( VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In other embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn is constant with the Fab heavy chain of the first Fab molecule regions share a carboxyl-terminal peptide bond (i.e., the first Fab molecule contains a swapped Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit ( VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ -CH2-CH3(-CH4)).

In some of these embodiments, the antibody further comprises: an exchanged Fab light chain polypeptide of the first Fab molecule, wherein the Fab heavy chain variable region of the first Fab molecule and the Fab light chain constant region of the first Fab molecule Shares a carboxyl-terminal peptide bond (VH ₍₁₎ - CL ₍₁₎ ) and shares a carboxyl-terminal peptide bond (VL ₍₂₎ - CL ₍₂₎ ) with the Fab light chain polypeptide of the second Fab molecule. In others of these embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the first Fab molecule, which in turn shares The Fab light chain polypeptide of the second Fab molecule shares a carboxyl-terminal peptide bond (VH ₍₁₎ -CL ₍₁₎ -VL ₍₂₎ -CL ₍₂₎ ); or a polypeptide, wherein the Fab light chain polypeptide of the second Fab molecule Shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL ₍₂₎ -CL ₍₂₎ -VH _{(1 )} -CL ₍₁₎ ) (where appropriate).

Antibodies according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxyl group with the Fc domain subunit terminal peptide bond (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)), and shares a carboxyl terminal peptide bond (VL ₍₃₎ -CL ₍₃₎ ) with the Fab light chain polypeptide of the third Fab molecule . In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

In some embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises an exchange-type Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH _{( 1)} -CL ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In other embodiments, the antibody comprises a polypeptide, wherein the Fab heavy chain of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn is constant with the Fab light chain of the first Fab molecule region shares a carboxyl-terminal peptide bond (i.e., the first Fab molecule contains a swapped Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH _{( 2)} -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ -CH2-CH3(-CH4)).

In some of these embodiments, the antibody further comprises: an exchanged Fab light chain polypeptide of the first Fab molecule, wherein the Fab light chain variable region of the first Fab molecule and the Fab heavy chain constant region of the first Fab molecule Shares a carboxyl-terminal peptide bond (VL ₍₁₎ -CH1 ₍₁₎ ) and shares a carboxyl-terminal peptide bond (VL ₍₂₎ -CL ₍₂₎ ) with the Fab light chain polypeptide of the second Fab molecule. In others of these embodiments, the antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule, which in turn shares The Fab light chain polypeptide of the second Fab molecule shares a carboxyl-terminal peptide bond (VL ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CL ₍₂₎ ); or a polypeptide, wherein the Fab light chain polypeptide of the second Fab molecule Shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL ₍₂₎ -CL ₍₂₎ -VH _{(1 )} -CL ₍₁₎ ) (where appropriate).

Antibodies according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxyl group with the Fc domain subunit terminal peptide bond (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)), and shares a carboxyl terminal peptide bond (VL ₍₃₎ -CL ₍₃₎ ) with the Fab light chain polypeptide of the third Fab molecule . In certain embodiments, the polypeptides are covalently linked, for example, via disulfide bonds.

In certain embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule The constant regions share a carboxyl-terminal peptide bond (i.e., the second Fab molecule contains a swapped Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region) (VH ₍₁₎ -CH1 ₍₁₎ -VL _{( 2)} -CH1 ₍₂₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ with the Fab light chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule.

In certain embodiments, the antibody comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchange type Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond (VL ₍₂₎ -CH1 ₍₂₎ -VH _{( 1)} -CH1 ₍₁₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ with the Fab light chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule.

In certain embodiments, the antibody comprises a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchange type Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ -VH _{( 1)} -CH1 ₍₁₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL ₍₂₎ -CH1 ₍₂₎ with the Fab heavy chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule.

In certain embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains an exchanged Fab heavy chain in which the heavy chain variable region is replaced by a light chain Variable region replacement) (VH ₍₃₎ -CH1 ₍₃₎ -VH ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CH1 ₍₂₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ with the Fab light chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares the Fab heavy chain variable region of the second Fab molecule Shares a carboxy-terminal peptide bond, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a swapped Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region Replacement) (VH ₍₃₎ -CH1 ₍₃₎ -VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL ₍₂₎ -CH1 ₍₂₎ with the Fab heavy chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, the antibody comprises a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchange Type Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares the carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares the Fab heavy chain of the third Fab molecule Carboxyl terminal peptide bond (VL ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₃₎ -CH1 ₍₃₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ with the Fab light chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, the antibody comprises a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchange Type Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the third Fab molecule Peptide bond (VH ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₃₎ -CH1 ₍₃₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL ₍₂₎ -CH1 ₍₂₎ with the Fab heavy chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule The constant region shares a carboxyl-terminal peptide bond (i.e., the second Fab molecule contains a swapped Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn is compatible with the Fab light chain of the third Fab molecule. The variable region shares a carboxyl-terminal peptide bond, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule contains an exchanged Fab heavy chain in which the heavy chain variable region is Chain variable region replacement) (VH ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CH1 ₍₂₎ -VL ₍₃₎ -CH1 ₍₃₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ with the Fab light chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide, wherein the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond (VH ₍₃₎ -CL ₍₃₎₎ with the Fab light chain constant region of the third Fab molecule. ).

In certain embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain of the second Fab molecule The constant regions share a carboxyl-terminal peptide bond (i.e., the second Fab molecule contains a swapped Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the third Fab molecule Shares a carboxy-terminal peptide bond, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule contains a swapped Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region Replace) (VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ -VH ₍₃₎ -CL ₍₃₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL ₍₂₎ -CH1 ₍₂₎ with the Fab heavy chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide, wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond (VL ₍₃₎ -CH1 ₍₃₎₎ with the Fab heavy chain constant region of the third Fab molecule. ).

In certain embodiments, the antibody comprises a polypeptide, wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises an exchange Type Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule The heavy chain constant region shares a carboxyl-terminal peptide bond (i.e., the second Fab molecule contains a swapped Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares the Fab heavy chain with the first Fab molecule. The chains share a carboxyl-terminal peptide bond (VL ₍₃₎ -CH1 ₍₃₎ -VL ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ with the Fab light chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide, wherein the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond (VH ₍₃₎ -CL ₍₃₎₎ with the Fab light chain constant region of the third Fab molecule. ).

In certain embodiments, the antibody comprises a polypeptide, wherein the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises an exchange Type Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain of the second Fab molecule The constant regions share a carboxyl-terminal peptide bond (i.e., the second Fab molecule contains a swapped Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxyl-terminus with the Fab heavy chain of the first Fab molecule Peptide bond (VH ₍₃₎ -CL ₍₃₎ -VH ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ ). In some embodiments, the antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL ₍₂₎ -CH1 ₍₂₎ with the Fab heavy chain constant region of the second Fab molecule ₎ ), and shares the carboxyl terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ ) with the Fab light chain polypeptide of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide, wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond (VL ₍₃₎ -CH1 ₍₃₎₎ with the Fab heavy chain constant region of the third Fab molecule. ).

According to any of the above embodiments, components of the antibody (e.g., Fab molecule, Fc domain) may be fused directly or through various linkers, in particular through the inclusion of one or more amino acids as described herein or as is known in the art. Typically about 2 to 20 amino acids) are fused to a peptide linker. Suitable non-immunogenic peptide linkers include, for example, (G ₄ S) _n (SEQ ID NO: 21), (SG ₄ ) _n (SEQ ID NO: 22) or G ₄ (SG ₄ ) _n (SEQ ID NO: 23) Peptide linker, where n is usually an integer from 1 to 10, usually from 2 to 4. 2. Fc domain

Anti-CD20/anti-CD3 bispecific antibodies may comprise an Fc domain consisting of a pair of polypeptide chains that comprise the heavy chain domain of the antibody molecule. For example, the Fc domain of the immunoglobulin G (IgG) molecule is a dimer, with each subunit containing the CH2 and CH3 IgG heavy chain constant domains. Two subunits of the Fc domain are able to stably associate with each other.

In one embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an _IgG1 Fc domain. In another embodiment, the Fc domain is an _IgG4 Fc domain. In a more specific embodiment, the Fc domain is an _IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), specifically the amino acid substitution S228P. This amino acid substitution reduces Fab arm exchange of IgG ₄ antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another specific embodiment, the Fc domain is a human Fc domain. (i) Fc domain modifications that promote heterodimerization

Anti-CD20/anti-CD3 bispecific antibodies can comprise different components (e.g., antigen-binding domains) that can be fused to one or the other of the two subunits of the Fc domain, whereby the two subunits of the Fc domain Units are usually contained in two non-identical polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. To improve the yield and purity of the recombinant production of such antibodies, it would be advantageous to introduce modifications into the Fc domain of the antibody that promote association of the desired polypeptide.

Accordingly, in certain embodiments, the Fc domain includes modifications that promote association of the first unit and the second unit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

Several methods of modifying the CH3 domain of the Fc domain to enhance heterodimerization are well disclosed in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009 /089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all of these approaches, the CH3 domain of the first unit of the Fc domain and the CH3 domain of the second unit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or containing the CH3 domain heavy chain) is no longer able to homodimerize with itself, but is forced to heterodimerize with other complementary engineered CH3 domains (making the first CH3 domain and the second CH3 domain heterologous Dimerize and do not form homodimers between the two first CH3 domains or the two second CH3 domains). These different approaches to improve heavy chain heterodimerization are considered to be related to heavy chain-light chain modifications (e.g., variable or constant region exchange/replacement in the Fab arms, or introduction in the CH1/CL interface). Different options for combining substituents with oppositely charged charged amino acids) that reduce light chain mispairing and Bence Jones type by-products.

In a specific embodiment, the modification that promotes the association of the first unit and the second unit of the Fc domain is a so-called "pestle" modification, which includes a "pestle" in one of the two subunits of the Fc domain. ” modification and the “mortar” modification of the other of the two subunits of the Fc domain.

"Pest and mortar" technology is disclosed in, for example: US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng . 9, 617-621 (1996) and Carter, J Immunol Meth . 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("pestle") at the interface of the first polypeptide and a corresponding cavity ("mortar") at the interface of the second polypeptide so that the protrusion can be positioned at in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing smaller amino acid side chains at the first polypeptide interface with larger side chains (eg, tyrosine or tryptophan). By replacing a larger amino acid side chain with a smaller amino acid side chain (such as alanine or threonine), a complementary cavity of the same or similar size as the protrusion is formed in the interface of the second polypeptide .

Accordingly, in a specific embodiment, in the CH3 domain of the first unit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, such that in the first A protrusion is generated in the CH3 domain of the second subunit, which can be positioned in the cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residue Substitution of amino acid residues with smaller side chain volumes creates a cavity within the CH3 domain of the second unit into which protrusions within the CH3 domain of the first unit can be positioned.

Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

Preferably, the amino acid residue with smaller side chain volume is selected from the group consisting of: alanine (A), serine (S), threonine (T) and valine (V).

Protrusions and cavities can be prepared by altering the nucleic acid encoding the polypeptide (eg, by site-specific mutations or by peptide synthesis).

In a specific embodiment, in the CH3 domain of the first unit ("subunit") of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the Fc domain In the CH3 domain of the second subunit ("mortar" subunit), the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment, additionally in the second subunit of the Fc domain, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced It is alanine residue (L368A) (EU number).

In yet further embodiments, additionally in the first unit of the Fc domain, the serine residue at position 354 is replaced with a cysteine residue (S354C) or a glutamic acid at position 356 The residue was replaced with a cysteine residue (E356C), and additionally in the second subunit of the Fc domain, the tyrosine residue at position 349 was replaced with a cysteine residue (Y349C) ( EU number). Introduction of these two cysteine residues results in the formation of a disulfide bond between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a specific embodiment, the first unit of the Fc domain includes amino acid substitutions S354C and T366W, and the second unit of the Fc domain includes amino acid substitutions Y349C, T366S, L368A, and Y407V (EU numbering).

In certain embodiments, the CD3 antigen-binding portion disclosed herein is fused to the first unit of the Fc domain (including the "bandle" modification). Without wishing to be bound by theory, fusion of the CD3 antigen-binding portion with the pestle-containing subunit of the Fc domain will (further) minimize the generation of bispecific antibodies containing two CD3 antigen-binding portions (two pestle-containing polypeptides three-dimensional conflict).

Other techniques for CH3 modification to enhance heterodimerization are understood as alternatives and are described for example in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007 /147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment, the heterodimerization method disclosed in EP 1870459 A1 is used instead. The method is based on the introduction of oppositely charged amino acids at specific amino acid positions at the CH3/CH3 domain interface between two subunits of the Fc domain. A preferred embodiment is the amino acid mutations R409D and K370E in one of the two CH3 domains (of the Fc domain); and the amino acid mutations D399K and E357K in the other of the two CH3 domains of the Fc domain ( EU number).

In another embodiment, the anti-CD20/anti-CD3 bispecific antibody may comprise the amino acid mutation T366W in the CH3 domain of the first unit of the Fc domain and the amine group in the CH3 domain of the second unit of the Fc domain The acid mutations T366S, L368A and Y407V, and in addition, the amino acid mutations R409D and K370E in the CH3 domain of the first unit of the Fc domain and the amino acid mutations D399K and K370E in the CH3 domain of the second unit of the Fc domain E357K (EU number).

In another embodiment, an anti-CD20/anti-CD3 bispecific antibody can comprise the amino acid mutations S354C and T366W in the CH3 domain of the first unit of the Fc domain and the amino acid mutations S354C and T366W in the CH3 domain of the second unit of the Fc domain. Amino acid mutations Y349C, T366S, L368A and Y407V, or the antibody contains amino acid mutations Y349C and T366W in the CH3 domain of the first unit of the Fc domain and amino acids in the CH3 domain of the second unit of the Fc domain Mutations S354C, T366S, L368A and Y407V, and additionally, the amino acid mutations R409D and K370E in the CH3 domain of the first unit of the Fc domain and the amino acid mutation D399K in the CH3 domain of the second unit of the Fc domain and E357K (all EU numbers).

In one embodiment, the heterodimerization method described in WO 2013/157953 may be used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K and the second CH3 domain contains the amino acid mutation L351D (EU numbering). In another embodiment, the first CH3 domain further comprises the amino acid mutation L351K. In a further embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (EU numbering).

In one embodiment, the heterodimerization method described in WO 2012/058768 may be used instead. In one embodiment, the first CH3 domain includes the amino acid mutations L351Y, Y407A, and the second CH3 domain includes the amino acid mutations T366A and K409F. In a further embodiment, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390 or K392, the mutation being selected from, for example: (a) T411N, T411R, T411Q, T411K, T411D , T411E or T411W; (b) D399R, D399W, D399Y or D399K; (c) S400E, S400D, S400R or S400K; (d) F405I, F405M, F405T, F405S, F405V or F405W; (e) N390R, N390 K or N390D ; or (f) K392V, K392M, K392R, K392L, K392F or K392E (EU number). In a further embodiment, the first CH3 domain includes the amino acid mutations L351Y and Y407A, and the second CH3 domain includes the amino acid mutations T366V and K409F. In a further embodiment, the first CH3 domain includes the amino acid mutation Y407A, and the second CH3 domain includes the amino acid mutations T366A and K409F. In a further embodiment, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (EU numbering).

In one embodiment, the heterodimerization method disclosed in WO 2011/143545 may be used instead, for example, amino acid modification at a position selected from 368 and 409 (EU numbering).

In one embodiment, the heterodimerization method described in WO 2011/090762 can be used instead, which method also uses the "mortar and pestle" technique described above. In one embodiment, the first CH3 domain contains the amino acid mutation T366W and the second CH3 domain contains the amino acid mutation Y407A. In one embodiment, the first CH3 domain contains the amino acid mutation T366Y and the second CH3 domain contains the amino acid mutation Y407T (EU numbering).

In one embodiment, the anti-CD20/anti-CD3 bispecific antibody or the Fc domain of the anti-CD20/anti-CD3 bispecific antibody belongs to the IgG ₂ subclass, and heterodimerization as described in WO 2010/129304 can be used Way.

In an alternative embodiment, modifications that promote association of the first and second units of the Fc domain include modifications that mediate electrostatic steering, such as disclosed in PCT Publication WO 2009/089004. Typically, this approach involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, thereby rendering homodimer formation electrostatically unfavorable but heterologous. Dimerization is electrostatically favorable. In one such embodiment, the first CH3 domain includes a negatively charged amino acid substitution of K392 and N392 (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain contains a positively charged amino acid substitution of D399, E356, D356 or E357 (such as lysine (K) or arginine (R), preferably D399K , E356K, D356K or E357K and preferably D399K and E356K). In further embodiments, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (such as glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In further embodiments, the first CH3 domain further or alternatively comprises a negatively charged amino acid (such as glutamic acid (E) or aspartic acid (D)) to the amine group of K439 and/or K370 Acid substitution (EU number).

In yet another embodiment, the heterodimerization method described in WO 2007/147901 may be used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (EU numbering).

In yet another embodiment, the heterodimerization method disclosed in WO 2007/110205 can be used.

In one embodiment, the first unit of the Fc domain includes amino acid substitutions K392D and K409D, and the second unit of the Fc domain includes amino acid substitutions D356K and D399K (EU numbering). (ii) Fc domain modifications that reduce Fc receptor binding and / or effector function

The Fc domain confers favorable pharmacokinetic properties to antibodies (e.g., anti-CD20/anti-CD3 bispecifics), including a long serum half-life, which facilitates good accumulation in target tissues and favorable tissue-to-blood distribution ratios . At the same time, however, this may result in the antibody being undesirably targeted to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Additionally, coactivation of the Fc receptor signaling pathway may result in cytokine release, which, when combined with other immunostimulatory properties that antibodies may possess and the long half-life of the antibody, results in the release of cytokine receptors following systemic administration. Overactivation and serious side effects.

Accordingly, in certain embodiments, the Fc domain of an anti-CD20/anti-CD3 bispecific antibody exhibits reduced binding affinity for Fc receptors and/or reduced effector function compared to the native _IgGi Fc domain. In one such embodiment, the Fc domain (or a molecule comprising the Fc domain, e.g., an antibody) exhibits less than 50% performance compared to a native _IgG1 Fc domain (or a corresponding molecule comprising a native _IgG1 Fc domain), Preferably less than 20%, more preferably less than 10% and most preferably less than 5% binding affinity for the Fc receptor, and/or with the native _IgG1 Fc domain (or with the native _IgG1 Fc domain) exhibit an effector function of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the corresponding molecule). In one embodiment, the Fc domain (or a molecule comprising the Fc domain, such as an antibody) does not substantially bind to Fc receptors and/or induce effector functions. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In a specific embodiment, the effect function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the nascent Fc receptor (FcRn) compared to the native _IgGi Fc domain. When the Fc domain (or a molecule comprising such an Fc domain, e.g., an antibody) exhibits greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90%, a native _IgG1 Fc domain (or an _IgG1 Fc domain-comprising When the corresponding molecule has binding affinity for FcRn, it achieves substantially similar binding to FcRn.

In certain embodiments, an engineered Fc domain has reduced binding affinity for an Fc receptor and/or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain contains one or more amino acid mutations that reduce the binding affinity and/or effector function of the Fc domain for Fc receptors. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the amino acid for the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain for the Fc receptor by at least 10-fold , at least 20 times or even at least 50 times. In some embodiments, a molecule (e.g., an antibody) comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5 % binding affinity to Fc receptors. In a specific embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to the complementary component, i.e., the specific binding affinity to C1q, is also reduced. In one embodiment, binding affinity to nascent Fc receptor (FcRn) is not reduced. When the Fc domain (or a molecule comprising the Fc domain, such as an antibody) exhibits greater than about 70% of the binding affinity of the non-engineered form of the Fc domain (or a corresponding molecule comprising the non-engineered form of the Fc domain) to the FcRn, Substantially similar binding to FcRn is achieved, that is, the binding affinity of the Fc domain to the receptor is maintained. An Fc domain or a molecule (e.g., an antibody) containing an Fc domain may exhibit such affinities of greater than about 80% and even greater than about 90%. In certain embodiments, an Fc domain is engineered to have a mutation that has reduced effector function compared to a non-engineered Fc domain. Reduced effector functions may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), Reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells , reduce direct signaling-induced apoptosis, reduce cross-linking of target-binding antibodies, reduce dendritic cell maturation, or reduce T cell priming. In one embodiment, the reduced effector function is selected from one or more of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the reduced effect function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by an unmodified Fc domain (or a corresponding molecule comprising an unmodified Fc domain).

In one embodiment, amino acid mutations that reduce the binding affinity and/or effector function of the Fc domain to the Fc receptor are amino acid substitutions. In one embodiment, the Fc domain contains an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331 and P329 (EU numbering). In a more specific embodiment, the Fc domain includes an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (EU numbering). In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A (EU numbering). In one such embodiment, the Fc domain is an _IgG1 Fc domain, particularly a human _IgG1 Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, especially P329G (EU number). In one embodiment, the Fc domain comprises an amino acid substitution at position P329, and a further amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297 and P331 (EU numbering). In more specific embodiments, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In certain embodiments, the Fc domain contains amino acid substitutions at positions P329, L234 and L235 (EU numbering). In a more specific embodiment, the Fc domain includes the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an _IgG1 Fc domain, particularly a human _IgG1 Fc domain. The amino acid substituted "P329G LALA" combination almost completely eliminates Fcγ receptor (as well as complement) binding of the human _IgG1 Fc domain, as described in PCT Publication No. WO 2012/130831, the entire text of which is incorporated herein by reference. WO 2012/130831 also describes methods for preparing such mutant Fc domains and determining their properties (eg Fc receptor binding or effector function).

IgG ₄ antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG ₁ antibodies. Thus, in some embodiments, the Fc domain is an _IgG4Fc domain, specifically a human _IgG4Fc domain. In one embodiment, the IgG ₄ Fc domain contains an amino acid substitution at position S228, specifically amino acid substitution S228P (EU numbering). To further reduce its binding affinity to the Fc receptor and/or its effector function, in one embodiment, the IgG ₄ Fc domain contains an amino acid substitution at position L235, specifically the amino acid substitution L235E (EU number). In another embodiment, the IgG ₄ Fc domain comprises an amino acid substitution at position P329, specifically amino acid substitution P329G (EU numbering). In a specific embodiment, the IgG ₄ Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (EU numbering). These _IgG4 Fc domain variants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, which is incorporated herein by reference in its entirety.

In particular embodiments, an Fc domain that exhibits reduced binding affinity to an Fc receptor and/or reduced effector function compared to a native _IgG1 Fc domain is one that includes the amino acid substitutions L234A, L235A, and optionally The human IgG ₁ Fc domain of P329G may be a human IgG ₄ Fc domain containing the amino acid substitutions S228P, L235E and optionally P329G (EU numbering).

In certain embodiments, N-glycosylation of the Fc domain is eliminated. In one such embodiment, the Fc domain contains an amino acid mutation at position N297, specifically asparagine is replaced with alanine (N297A) or aspartic acid (N297D) or glycine (N297G ) amino acid substitution (EU number).

In addition to the Fc domains disclosed above and in PCT Publication No. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector functions also include those at Fc domain residues 238, 265, 269, and 270 , 297, 327 and 329 have substitutions for one or more of them (US Patent No. 6,737,056) (EU number). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc variants in which residues 265 and 297 Substituted by alanine (U.S. Patent No. 7,332,581).

Variant Fc domains can be prepared by amino acid deletions, substitutions, insertions or modifications using genetic or chemical methods known in the art. Genetic methods can include site-specific mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined by ELISA or by surface plasmon resonance (SPR) using standard instrumentation such as BIACORE® Instruments (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, the binding affinity of an Fc domain or an Fc domain-containing molecule for an Fc receptor can be assessed using cell lines known to express specific Fc receptors (e.g., human NK cells expressing FcγIIIa receptors).

The effector function of an Fc domain or a molecule containing an Fc domain (eg, an antibody) can be measured by methods known in the art. This article reveals suitable analytical methods for measuring ADCC. Other examples of in vitro assays for assessing ADCC activity of molecules of interest are disclosed in: U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA. 83, 7059-7063 (1986) and Hellstrom et al. Proc Natl Acad Sci USA .82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, nonradioactive assay methods may be used (see, e.g., ACTI™ Nonradioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® Nonradioactive Cytotoxicity Assay (Promega , Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, for example, the ADCC activity of the target molecule can be assessed in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, the binding of the Fc domain to complement components, specifically to Clq, is reduced. Thus, in some embodiments, wherein the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether an Fc domain or an Fc domain-containing molecule (eg, an antibody) binds C1q and therefore has CDC activity. See, for example, the C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, the CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103 , 2738-2743 (2004)). 3. Substitution, insertion and deletion

In some cases, anti-CD20/anti-CD3 bispecific antibody variants of the pharmaceutical compositions provided herein have one or more amino acid substitutions. Target sites for substitution mutagenesis include HVR and FR. Retaining substitutions are listed in Table 3 under the heading "Preferred Substitutions". More substantial changes are provided in Table 3 under the heading "Exemplary Substitutions" and are further described below with reference to the amino acid side chain class. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, eg, retained/improved antigen binding characteristics, reduced immunogenicity, or improved ADCC or CDC. Table 3. Exemplary and preferred amino acid substitutions original residue illustrative substitution better replacement Ala (A) Val;Leu;Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg gnc Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp;Gln Asp Gly(G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu;Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val;Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp;Phe;Thr;Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

Nonconservative substitutions require the exchange of a member of one of these classes for a member of the other class.

One type of substitution variant involves substituting the parent antibody (e.g., humanized or human antibody) one or more highly variable region residues. Typically, the resulting variants selected for further study will have modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity, reduced immunogenicity) and/or substantially retain the parental antibody. Certain biological properties of antibodies. Exemplary substitution variants are affinity matured antibodies, which can be conveniently produced, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, and variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

Changes (eg, substitutions) can be made in HVR to improve antibody affinity. Such modifications can occur in HVR "hot spots," i.e., residues encoded by codons that undergo high frequency mutations during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and or residues that contact the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, by Hoogenboom et al. Methods in Molecular Biology 178:1-37 (eds. O'Brien et al., Human Press, Totowa, NJ (2001)). In some examples of affinity maturation, diversity is introduced into variant genes selected for maturation by a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide site-directed mutagenesis). Then create a second library. The library is then screened to identify any antibody variants with the desired affinity. Another way to introduce diversity is the HVR directed method, in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified by, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

In some instances, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such modifications do not significantly reduce the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions as described herein) that do not substantially reduce binding affinity can be implemented in HVR. For example, such modifications may be outside the antigen-contacting residues in the HVR. In certain examples of the VH and VL sequence variants described above, each HVR is unchanged or includes no more than one, two, or three amino acid substitutions.

As described by Cunningham and Wells (1989) ( Science , 244:1081-1085), a useful method for identifying antibody residues or regions that may be mutagenic is called alanine scanning mutagenesis. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and used with neutral or negatively charged amino acids (e.g., alanine or polyalanine) substitution to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions, indicating good functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. These contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include the length of amine and/or carboxyl terminal fusions, from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include enzymes fused to the N- or C-terminus of the antibody (eg, for ADEPT) or peptides that increase the serum half-life of the antibody. 4. Glycosylation

In certain examples, the anti-CD20/anti-CD3 bispecific antibodies of the invention included in pharmaceutical compositions can be modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites in anti-CD20/anti-CD3 bispecific antibodies can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

When an antibody contains an Fc region, the carbohydrate to which it is linked can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides attached to Asn297 of the CH2 domain of the Fc region, often via N-bonding. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure . In some examples, oligosaccharides in antibodies are modified to produce antibody variants with certain improved properties.

In one example, an anti-CD20/anti-CD3 bispecific antibody variant has a carbohydrate structure lacking fucose linked (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies can range from 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. Fucose was determined by calculating the average fucose content in the Asn297 glycan relative to all sugar structures linked to Asn297 (e.g., complex, hybrid, and high mannose structures) measured by MALDI-TOF mass spectrometry ), for example, as disclosed in WO 2008/077546. Asn297 refers to the asparagine residue located near position 297 in the Fc region (EU numbering of the Fc region residue); however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., due to antibody There is a slight sequence change between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "afucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328 ; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 200 5/035778;WO 2005/053742 ; WO 2002/031140; Okazaki et al., J. Mol. Biol. 336:1239-1249, 2004; Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially in Example 11); and knockout cell lines, such as knockout of α-1,6-fucosyltransferase CHO cells expressing FUT8 (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al. , Biotechnol. Bioeng ., 94(4):680-688 (2006); and WO 2003/085107).

In view of the above, in some examples, pharmaceutical compositions of the invention comprise anti-CD20/anti-CD3 bispecific antibody variants that comprise glycosylation site mutations. In some examples, glycosylation site mutations reduce the effector function of the antibody. In some examples, glycosylation site mutations are substitution mutations. In some instances, the antibody contains a substitution mutation in the Fc region that reduces effector function. In some examples, substitution mutations are located at amino acid residues N297, L234, L235, and/or D265 (EU numbering). In some examples, the substitution mutation is selected from the group consisting of: N297G, N297A, L234A, L235A, D265A, and P329G. In some instances, the substitution mutation is located at amino acid residue N297. In a preferred example, the substitution mutation is N297A.

Anti-CD20/anti-CD3 bispecific antibody variants may contain bisected oligosaccharides, for example, in which a biantennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are disclosed, for example, in WO 2003/011878; US Patent No. 6,602,684; and US 2005/0123546. Other antibody variants contain at least one galactose residue on the oligosaccharide linked to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are disclosed, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764. 5. Antibody derivatives

In certain examples, the anti-CD20/anti-CD3 bispecific antibodies of the pharmaceutical compositions provided herein are further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, Poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylenated polyol (eg glycerin), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used in the specified conditions. The treatment below is moderate.

In another example, conjugates of antibodies and non-protein moieties can be selectively heated by exposure to radiation. In one example, the non-protein moieties are carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage ordinary cells but heat the non-protein portion to a temperature close to that at which the antibody-non-protein portion of the cell is killed. C. Recombinant production method

The anti-CD20/anti-CD3 bispecific antibodies (eg, anti-CD20/anti-CD3 TCB, eg, gaffetuzumab) of the pharmaceutical compositions of the present invention can be produced using recombinant methods and compositions, for example, as in U.S. Pat. No. 4,816,567 No. 1, the entirety of which is incorporated herein by reference.

For recombinant production of anti-CD20/anti-CD3 bispecific antibodies, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further selection and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of binding specifically to genes encoding antibody heavy and light chains).

Suitable host cells for the selection or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology , vol . 248 (ed. BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli .) After expression , the antibodies can be separated from the soluble fraction of the bacterial cell paste and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeasts) are also hosts for the selection or expression of suitable antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized." This results in the production of antibodies with partially or fully human glycosylation patterns. See: Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (disclosing PLANTIBODIES ^™ technology for producing antibodies in genetically modified plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines include: the monkey kidney CV1 line transformed with SV40 (COS-7); the human embryonic kidney line (as described in Graham et al., J. Gen Virol. 36:59 (1977) 293 or 293 cells); baby hamster kidney cells (BHK); mouse testicular Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells ( Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described by Mather et al., Annals NYAcad. Sci . 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, For example Y0, NS0 and Sp2/0. For a review of some mammalian host cell strains suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology , vol . 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 ( 2003). V. Treatment methods and uses

Pharmaceutical compositions containing the anti-CD20/anti-CD3 bispecific antibodies described herein can be formulated for use as medicaments for the treatment of various diseases and conditions. Accordingly, the present invention features methods for intravenously administering a pharmaceutical composition to an individual in need thereof, such as a subject suffering from a disease or condition such as cancer. The pharmaceutical compositions of the present invention may be used to treat or delay the progression of a cell proliferative disorder in an individual in need thereof (e.g., a human subject in need thereof) or to enhance the effectiveness of an individual suffering from a cell proliferative disorder (e.g., cancer). Immune Function.

In one aspect, the invention provides pharmaceutical compositions as described herein for treating or delaying the progression of a cell proliferative disorder. In one aspect, the present invention provides the use of a pharmaceutical composition described herein in the manufacture of a medicament for treating or slowing the progression of a cell proliferative disorder. In one aspect, the present invention provides a method of treating or delaying the progression of a cell proliferative disorder in an individual in need thereof, comprising administering to the individual a pharmaceutical composition as described herein.

In some embodiments, the cell proliferative disorder is a cancer that is non-Hodgkin's lymphoma (NHL). In some embodiments, NHL is selected from the group consisting of: non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma , B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, Burkitt-like lymphoma with 11q abnormality, features between diffuse large B-cell lymphoma and typical Characteristic B-cell lymphomas among Hodgkin's lymphomas, germinal center B-cell-like (GCB), diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, primary cutaneous filter Follicular center lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), Estam-Barr virus (EBV) positivity in the elderly DLBCL, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, in HHV8-associated multicentric Castleman's disease Caused by large B-cell lymphoma, B-cell leukemia, breast cancer, colorectal cancer, non-small cell lung cancer, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, neurological Glioblastoma, follicular lymphoma (FL), follicular neoplasm in situ, mantle cell lymphoma (MCL), mantle cell tumor in situ, acute myeloid leukemia (AML), marginal zone lymphoma (MZL) , small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prelymphocytic leukemia, splenic marginal zone lymphoma neoplasia, hairy cell leukemia, splenic lymphoma/leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma , mucosa-associated lymphoid tissue extranodal marginal zone lymphoma (MALT lymphoma), nodular marginal zone lymphoma, pediatric nodular marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granulomatosis, plasmablastic cell Lymphoma and primary effusion lymphoma. In specific embodiments, the cancer is germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML) ), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL) or Burkitt's lymphoma (BL).

In some embodiments, the cancer is selected from the group consisting of: breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC), multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer carcinoma, mesothelioma, and glioblastoma.

Anti-CD20/anti-CD3 bispecific antibodies can be formulated for administration to individuals at doses of 0.5 mg, 2.5 mg, 10 mg, or 30 mg.

For all methods and pharmaceutical compositions described herein, the anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) will be formulated, administered, and administered in a manner consistent with good medical practice. Medicines and administration. In this case, factors to be considered include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and the medical practitioner's experience other factors known. Anti-CD20/anti-CD3 bispecific antibodies (eg, anti-CD20/anti-CD3 TCB, eg, gaffetuzumab) need not be formulated with one or more agents currently used to prevent or treat all relevant disorders, as appropriate. The effective amount of such other agents depends on the amount of anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab) present in the formulation, the type of disorder or treatment, and Other factors as discussed above. Anti-CD20/anti-CD3 bispecific antibodies (eg, anti-CD20/anti-CD3 TCB, eg, gaffetuzumab) can be appropriately administered to patients over a series of treatments. VI. Finished products

In another aspect of the invention, articles are provided containing materials useful in treating, preventing and/or diagnosing the disorders described above. Finished products include containers and labels or package inserts on or associated with the containers. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container contains a pharmaceutical composition, either by itself or in combination with another composition effective in treating, preventing and/or diagnosing a condition, and may have a sterile access port (e.g., the container may have an intravenous solution bag or a transdermal injection needle perforated stopper vial). At least one active agent in the composition is an anti-CD20/anti-CD3 bispecific antibody (e.g., anti-CD20/anti-CD3 TCB, e.g., gaffetuzumab), as described herein. The label or package insert indicates that the composition is for use in treating a selected condition (e.g., cancer), and further includes information regarding at least one of the dosage regimens described herein.

Pharmaceutical compositions may be provided in volumes ranging from 1 ml to 100 ml (e.g., 1 ml to 5 ml, 5 ml to 10 ml, 10 ml to 15 ml, 15 ml to 20 ml, 20 ml to 25 ml, 25 ml to 30 ml). ml, 30 ml to 40 ml, 40 ml to 50 ml, 50 ml to 60 ml, 60 ml to 70 ml, 70 ml to 80 ml, 80 ml to 90 ml, or 90 ml to 100 ml, for example, about 5 ml, About 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 40 ml, about 50 ml, about 60 ml, about 70 ml, about 80 ml, about 90 ml, or about 100 ml) in the container.

In some embodiments, the container is a stainless steel container or a nickel steel alloy container (e.g., HASTELLOY®), such as a can, a microcan, a canister, a can, etc. In some examples, the pharmaceutical composition in such a container is a drug substance (DS), which may be further diluted prior to use, e.g., to become a drug product (DP) (e.g., in a final vial configuration). Alternatively, the pharmaceutical composition in the container is DP. In some embodiments, the DP is in a container such as an IV bag or syringe (eg, for delivery via a syringe pump).

In some embodiments, the article of manufacture includes a volume of about 1 ml or greater, such as about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, About 9 ml, about 10 ml, about 11 ml, about 12 ml, about 13 ml, about 14 ml, about 15 ml, about 16 ml, about 17 ml, about 18 ml, about 19 ml, about 20 ml, about 25 ml, approximately 30 ml, approximately 35 ml, approximately 40 ml, approximately 50 ml or larger vials. In some embodiments, the container is a vial with a volume of approximately 10 ml. In some embodiments, the vial is for single use. In some embodiments, the vial contains about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg or more anti-CD20/anti-CD3 bispecific antibodies (eg, anti-CD20/anti-CD3 TCB, eg, gaffetuzumab). In some embodiments, the container closure system includes one or more, or all, of a glass vial, a stopper, and a cap.

Additionally, the article of manufacture may comprise (a) a first container containing a pharmaceutical composition therein, wherein the composition comprises an anti-CD20/anti-CD3 bispecific antibody described herein (e.g., anti-CD20/anti-CD3 TCB, e.g., Filtuzumab); and (b) a second container containing a pharmaceutical composition therein, wherein the composition includes another cytotoxic agent or other therapeutic agent. Alternatively or additionally, the finished article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. From a commercial and user perspective, it can further contain other materials, including other buffers, diluents, filters, needles and syringes.

Another aspect of the invention relates to the invention as described above. Example

Some embodiments of the technology described herein may be defined according to any of the following numbered examples: I. A liquid pharmaceutical composition comprising: about 1 to 25 mg/ml of an anti-CD20/anti-CD3 bispecific antibody; about 10 to 50 mM of buffer; about ≥200 mM of tonicity agent; about 0 to 15 mM methionine; and about ≥0.2 mg/ml of surfactant pH in the range of about 5.0 to about 6.0, where anti- The CD20/anti-CD3 bispecific antibody includes: a) at least one antigen-binding domain that specifically binds to CD20, which includes a heavy chain variable region, which includes: (i) HVR-H1, which includes SEQ ID NO: 1 The amino acid sequence; (ii) HVR-H2, which includes the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which includes the amino acid sequence of SEQ ID NO: 3; and the light chain may A variable region comprising: (i) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which includes the amino acid sequence of SEQ ID NO: 6; and b) at least one antigen-binding domain that specifically binds to CD3, which includes a heavy chain variable region, which includes: (i) HVR-H1, It includes the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which includes the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which includes the amine of SEQ ID NO: 11 and a light chain variable region, which includes: (i) HVR-L1, which includes the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which includes the amine of SEQ ID NO: 13 and (iii) HVR-L3, which includes the amino acid sequence of SEQ ID NO: 14. II. The liquid pharmaceutical composition of embodiment I, wherein the anti-CD20/anti-CD3 bispecific antibody concentration is in the range of about 1 to 5 mg/ml. III. The liquid pharmaceutical composition of any one of the preceding embodiments, wherein the anti-CD20/anti-CD3 bispecific antibody concentration is in the range of about 0.9 to 1.1 mg/ml. IV. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the anti-CD20/anti-CD3 bispecific antibody concentration is about 1 mg/ml. V. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the anti-CD20/anti-CD3 bispecific antibody includes: a) at least one antigen-binding domain that specifically binds to CD20, which includes SEQ ID NO: 7 The heavy chain variable region sequence and the light chain variable region sequence of SEQ ID NO: 8, and b) at least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and The light chain variable region sequence of SEQ ID NO: 16. VI. The liquid pharmaceutical composition of any one of the preceding embodiments, wherein the anti-CD20/anti-CD3 bispecific antibody comprises: a) a first Fab molecule that specifically binds to CD3, specifically CD3 epsilon; and wherein The variable domains VL and VH of the Fab light chain and Fab heavy chain replace each other; b) the second Fab and the third Fab molecule, which specifically bind to CD20, wherein in the constant domain CL of the second Fab and the third Fab molecule , the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R), specifically Substituted by arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the second and third Fab molecules, the amino acid at position 147 is substituted by glutamic acid (E) (EU numbering ), and the amino acid at position 213 is replaced by glutamic acid (E) (EU numbering); and c) c) Fc domain, which consists of a first unit and a second unit capable of stable association. VII. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the anti-CD20/anti-CD3 bispecific antibody is gaffetuzumab. VIII. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the buffer is histidine buffer, optionally histidine HCl buffer. IX. The liquid pharmaceutical composition of any one of the preceding embodiments, wherein the buffer is at a concentration of about 15 to 25 mM. X. The liquid pharmaceutical composition of any one of the preceding embodiments, wherein the buffer is at a concentration of about 20 mM. XI. The liquid pharmaceutical composition of any one of the preceding embodiments, wherein the buffer provides a pH of about 5.2 to about 5.8. XII. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the tonicity agent is selected from the group of salts, sugars and amino acids. XIII. The liquid pharmaceutical composition of Embodiment XII, wherein the tonicity agent is sucrose or sodium chloride. XIV. The liquid pharmaceutical composition of Embodiment XIII, wherein the tonicity agent is sucrose at a concentration of about 200 mM or higher. XV. The liquid pharmaceutical composition of embodiment XIII or XIV, wherein the tonicity agent is sucrose at a concentration of about 200 mM to 280 mM. XVI. The liquid pharmaceutical composition of any one of embodiments XIII to XV, wherein the tonicity agent is sucrose at a concentration of about 240 mM. XVII. The liquid pharmaceutical composition of any one of the preceding embodiments, wherein methionine is at a concentration of about 5 to 15 mM. XVIII. The liquid pharmaceutical composition of embodiment XVII, wherein methionine is at a concentration of about 10 mM. XIX. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the surfactant is at a concentration of about 0.2 to 0.8 mg/ml. XX. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the surfactant is polysorbate 20 or poloxamer 188. XXI. The liquid pharmaceutical composition of embodiment XX, wherein the surfactant is polysorbate 20 at a concentration of 0.2 to 0.8 mg/ml. XXII. The liquid pharmaceutical composition of embodiment XXI, wherein the surfactant is polysorbate 20 at a concentration of about 0.5 mg/ml. XXIII. The liquid pharmaceutical composition according to any one of the preceding embodiments, comprising: about 1 to 5 mg/ml of anti-CD20/anti-CD3 bispecific antibody; about 15 to 25 mM histidine buffer; about 200 to 280 mM sucrose; about 0 to 15 mM methionine; and about 0.2 to 0.8 mg/ml of PS20 pH of about 5 to about 6. XXIV. The liquid pharmaceutical composition according to any one of the preceding embodiments, comprising: about 1 mg/ml greffitumab; about 20 mM histidine buffer; about 240 mM sucrose; about 10 mM formazan. Thiamine; and about 0.5 mg/ml of PS20 with a pH of about 5.5. XXV. Use of a liquid pharmaceutical composition according to any one of the preceding embodiments for the preparation of a medicament for the treatment of cell proliferative disorders. XXVI. The pharmaceutical composition according to any one of embodiments I to XXIV, which is used to treat or delay the progression of a cell proliferative disorder in an individual in need. XXVII. A method of treating or delaying the progression of a cell proliferative disorder in an individual in need thereof, the method comprising administering to the individual an effective amount of a liquid pharmaceutical composition such as any one of Embodiments I to XXIV. XXVIII. The use, liquid pharmaceutical composition or method of any one of embodiments XXV to XXVII, wherein the cell proliferative disorder is cancer. XXIX. The invention as described above. Example

The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in view of the general description given above. Example 1 : Computer simulation analysis of gaffetuzumab

RO7082859 / Gerfitumab is a T cell bispecific humanized monoclonal antibody (TCB) that binds to human CD20 on tumor cells and human CD3 of the T cell receptor complex (TCR) on T cells ε unit (CD3ε) binding. It consists of two different heavy chains and two different light chains. Point mutations ("pestles") in the CH3 domain promote the assembly of two different heavy chains. Swapping of the VH and VL domains in the CD3-binding Fab (the "CrossMab approach") and point mutations in the CH and CL domains of the CD20-binding Fab ("charged variants") promote the correct assembly of two different light chains and the corresponding heavy chain. The "Pestlet" mutation consists of amine exchanges Y349C, T366S, L368A and Y407V in heavy chain HC1 and amine exchanges S354C and T366W in heavy chain HC2 (Kabat EU index number). The "charged variant" mutations consist of amino acid exchanges E123R and Q124K (Kabat numbering) in the light chain LC2 and K147E and K213E (Kabat EU indexing number) in the heavy chains HC1 and HC2.

Binding to human CD20 occurs in a high-affinity, bivalent binding mode, whereas binding to CD3ε is monovalent and low-affinity. RO7082859 is a human IgG1 with modifications in its Fc region ("PG LALA" mutation) that can eliminate its binding to Fcγ receptors (FcγR) in vitro and prevent FcγR-mediated co-activation of innate immune effector cells. There is no change in the functional binding of natural killer (NK) cells, monocytes/macrophages and neutrophils to FcRn (nascent Fc receptor). The "PG LALA" mutation consists of amino acid exchanges P329G, L234A and L235A in heavy chain HC1 and heavy chain HC2 ("PG LALA", Kabat EU index number).

The recombinant antibody is produced in CHO cells and consists of two heavy chains (449 and 674 amino acid residues, respectively) and three light chains (232 and 219 (two copies) amino acid residues, respectively). , arranged in an asymmetric configuration, as shown in Figure 2.

Summary of active hotspots

For the CD3-binding portion of the molecule, computer simulation predictions indicate that the heavy chain CDR3 has two Asn residues that are prone to degradation and one exposed Trp residue. In stress experiments over 14 days, no major changes in target-binding activity were observed after incubation at pH 6.0, but a strong loss of target-binding activity was observed after incubation at physiological pH (PBS pH 7.4, data not shown). Example 2 : Development of GLP Toxicity and Entering Human Studies of Gefituzumab Formulation

Filtering was performed according to the scheme shown in Table 4. During screening, formulations were exposed to the following conditions: 3 and 6 weeks of storage (at 5°C, 25°C, and 40°C), 1 week of shaking at 5°C and 25°C, and freeze/thaw (F/T) pressure (5 cycle). Selected formulations were then followed for up to 52 weeks. Table 4 : Adaptation platform screening study design and formulation codes Preparations Gerfituzumab protein concentration (mg/ml) Buffer pH Excipient 1 Excipient 2 surfactant F1 5 20mM His/His-Cl 5.5 240 mM sucrose 10 mM methionine 0.05 (w/v)% PS20 F2 5 20mM His/His-Cl 5.5 240 mM sucrose - 0.05 (w/v)% PS20 F3 5 20mM His/His-Cl 5.5 240 mM sucrose 10 mM methionine 0.05 (w/v)% Px188 F4 5 20mM His/His-Cl 5.5 240 mM sucrose - 0.05 (w/v)% Px188 F5 5 20mM His/His-Cl 6.0 240 mM sucrose 10 mM methionine 0.05 (w/v)% PS20

After 6 weeks of storage at 5°C, 25°C and 40°C, all formulations showed no significant changes in most of the physical properties tested (i.e. visible and invisible particles, color, turbidity, pH and protein content). CE-SDS (capillary electrophoresis sodium dodecyl sulfate) data is not shown because it is not important for the selection.

Visible particle analysis by the Seidenader method showed that no visible particles were formed in either formulation under all storage conditions. Low particle counts not visible to the naked eye (not shown). Under mechanical stress conditions, F2-F5 showed many particles at both 5 and 25°C. F1 was particle-free in both conditions. Using EP and Optima, all compositions were almost free of particles (0 particles) except F3 and F4 (both with P188) which showed particles but below the limit (not shown). Visually invisible particles were significantly worse in F3 (P188 + Met) than in F4 (P188) with shaking at 5°C, with all other formulations having similar counts in each condition (not shown).

After 6 weeks, there was no significant change in turbidity or color of all formulations under all conditions. The surfactant content was stable at 5 and 25°C, and also at 40°C for the two formulations containing P188 (F3, F4). A loss in surfactant content was observed at 40°C for all active formulations containing PS20 (F1, F2 and F5), regardless of whether the formulations contained methionine.

The beneficial effect of methionine was only seen in the placebo formulation containing PS20, where only P2 showed a decrease in PS content at 40°C (Fig. 3). Biochemical characterization revealed differences in the formulations only after storage at 40°C.

In size sieve chromatography (SEC), monomer losses were more pronounced for F2 and F5, which correlated with an increase in HMW (high molecular weight) area. It can be seen that new HMW species appear, only minor in F3 and F4, stronger in F1, and dominant in F2 and F5. LMW (low molecular weight) species were found to increase at approximately the same rate in all formulations (Figure 4). A similar trend can be observed in ion exchange chromatography (IEC), with an overall increase in basic peak area and an increase in acidic peak area that is more pronounced in F2 and F5 (Figure 5).

In summary, the data clearly exclude F2 and F5, and show that F1, F3, and F4 are equally stable, with no obvious bias toward any one of the three. Selected F1 (5 mg/ml greffitumab, 20 mM histidine/histidine HCl, pH 5.5, 240 mM sucrose, 10 mM methionine, 0.05% (w/v) PS20). A summary of all analytical results for F1 can be found in Figure 6 . Example 3 : GLP toxicity / human entry studies

BIACORE® combines

The above purity results are also reflected in the loss of CD20 binding of F2 and F5 at 40°C, as well as the strong loss of CD3 binding in up to 50% of these formulations compared with losses between 10 and 20% in the remaining formulations (Figure 7A and Figure 7B). Example 4 : Phase III and commercial formulation development studies

This example provides an overview of the drug development of gaffetuzumab formulations. As a result of this development, the gaffetuzumab drug product is available as a sterile liquid concentrate for use as an IV infusion solution. This drug product consists of 1 mg/ml greffitumab, 240 mM sucrose, 10 mM L-methionine, Composition of 0.5 mg/ml polysorbate 20, pH 5.5. Gefituzumab is the only active ingredient in medicinal substances and medicinal products. Formulation development studies determine that the dosage form and formulation are suitable for the intended use. The formulation is sufficiently robust to ensure the stability of the drug product during manufacture, storage, transportation and administration.

Formulations with higher protein concentrations (e.g., 5, 25, or 50 mg/ml gaffetuzumab) were tested but were not pursued due to PS20 degradation leading to visual invisibility and visible particle formation. The release of free fatty acids (lauric acid and myristic acid) with increasing protein concentration confirms that the underlying cause of the formation of invisible and visible particles is due to hydrolytic PS20 degradation.

A liquid dosage form was selected that would reduce handling steps while ensuring product quality during manufacturing and at the end of the drug product's shelf life.

The gaffetuzumab drug product will be marketed in two strengths and two vial configurations: 2.5 mg/vial in a 6-ml single-use glass vial and 10 mg/vial in a 15-ml single-use glass vial to match the required clinical doses of 2.5, 10 and 30 mg while minimizing product waste. For commercial drug product formulations, the concentration of gaffetuzumab was reduced to 1 mg/ml while maintaining the excipient composition unchanged.

Formulation development studies provide the basis for selecting appropriate dosage forms, protein concentrations, surfactant concentrations, buffer materials, solution pH, stabilizers, tonicity agents, and vial configurations for the drug product. Drug substance formulations are optimized to take into account facilities, dilution and storage considerations.

Choice of dosage form

Select a liquid dosage form that provides a concentrate of the solution for infusion that requires few processing steps while ensuring product quality during manufacturing and at the end of the drug product's shelf life.

Choice of protein concentration

A protein concentration of 5 mg/ml was selected for phase I and retained until phase III. Based on formulation development studies and updated clinical dosage requirements, a protein concentration of 1 mg/ml was subsequently selected as a commercial formulation.

Formulated with 20 mM L-histidine/L-histamine hydrochloride, 10 mM L-methionine, 240 mM D-sucrose, and 0.5 mg/ml polysorbate 20 (PS20), pH 5.5 The stability of the substance was tested at gaffetuzumab concentrations of 1 mg/ml, 5 mg/ml and 25 mg/ml in preparation for adapting the protein concentration to clinical needs. The purity and PS20 content of these formulations were evaluated by SE-HPLC and IE-HPLC after 104 weeks of storage at 2°C-8°C at the initial time point (T0), several intermediate time points, and at the end of the study. and visible/visually invisible particle formation were evaluated.

Throughout the study, purity determined by SE-HPLC and IE-HPLC was comparable between the 1 mg/ml and 5 mg/ml formulations (Figure 8A and Figure 8B). Invisible particle counts were also comparable. Additionally, the 1 mg/ml formulation did not exhibit PS20 degradation beyond method variability compared to the 5 mg/ml and 25 mg/ml formulations (Figure 12, see also below, Evaluation of Polysorbate 20 Degradation) . Based on these results and updated clinical dosing regimens of 2.5, 10, and 30 mg, the 1 mg/ml formulation was selected as the commercial formulation.

The concentration range of 0.9-1.1 mg/ml protein was further evaluated in a subsequent multivariable formulation robustness study (see Example 5, Formulation Robustness Study). This study confirmed acceptable stability behavior within this concentration range. Selection of pH , buffer, stabilizer and tonicity agent

Based on formulation development studies, a 20 mM L-histidine/L-histamine hydrochloride solution at pH 5.5 was selected as the buffer, combined with 10 mM L-methionine as a stabilizer and 240 mM D-sucrose as Tonicity agent for Phase I and reserved for Phase III and commercial formulations.

A study with 5 mg/ml gaffetuzumab was set up to test 20 mM L-histamine/L-histamine hydrochloride buffer with a pH range of 5.5 to 6.0 and an L-methionine content of 0 and 10mM. Additionally, 240 mM D-sucrose and 130 mM sodium chloride were compared.

The effects of pH and stabilizers were evaluated by SE-HPLC and IE-HPLC to assess the purity and visible/visible invisible particle formation of gaffetuzumab at the initial time point (T0) and after 6 weeks of storage at 40°C. The choice of tonicity agent was evaluated at initial time point (T0) and after 26 weeks of storage at 25°C by measuring SE-HPLC, IE-HPLC and determining visible/visually invisible particle formation. at pH 5.5 compared to the corresponding formulation without stabilizer addition or the 20 mM L-histidine/L-histamine hydrochloride buffer/10 mM L-methionine combination at pH 6 The combination of 20 mM L-histidine/L-histamine hydrochloride buffer with 10 mM L-methionine showed the lowest formation of high molecular weight species (HMWS) (Figure 9A) and charge variants. changes (Figure 9B). A concentration of L-histamine/L-histamine hydrochloride monohydrate of 20 mM was shown to be sufficient to maintain formulation pH during manufacture of the drug product and during storage of the drug substance and the drug product.

Based on the comparison between 240 mM D-sucrose and 130 mM sodium chloride, 240 mM D-sucrose was selected. The number of invisible particles was comparable between formulations. For formulations containing D-sucrose, no visible particle formation was observed after 26 weeks of storage at 25°C, whereas visible particles were observed for formulations containing NaCl (Figure 10). Surfactant selection

PS20 at a concentration of 0.5 mg/ml was selected for Phase I and retained for commercial formulation based on stability study results. Studies with 50 mg/ml gaffetuzumab in 20 mM L-histidine/L-histamine hydrochloride buffer, pH 5.5, with 10 mM L-methionine and 240 mM D-sucrose , to investigate the stabilizing effect of poloxamer 188 (P188) and PS20. P188 was tested at 0.5, 0.7 and 1.0 mg/ml; PS20 was tested at 0.1, 0.3 and 0.5 mg/ml.

The effect of the added surfactant was evaluated by SE-HPLC and IE-HPLC to assess the purity and visible/invisible particle formation of gaffetuzumab at the initial time point (T0) and after 7 days of shaking at 25°C.

Visible particle formation was observed for all P188 concentrations. It was thus ruled out as a suitable surfactant for gaffetuzumab (Fig. 11). After 7 days of shaking at 25°C, no visible particles were detected in formulations containing PS20 (Figure 11). A significant increase in HMWS and charge variants was observed for formulations containing 0.1 mg/ml PS20, whereas for formulations containing 0.3 mg/ml PS20, there was a significant increase in HMWS compared to formulations containing 0.5 mg/ml PS20 after 7 days of shaking at 25°C. A slight increase in HMWS and charge variant content was observed compared to the formulations (Figure 11). Visually invisible particle counts were comparable at different PS20 concentrations. For formulations containing 0.1 mg/ml PS20, a significant increase in HMWS and charge variants was observed, whereas for formulations containing 0.3 mg/ml PS20, there was a significant increase in HMWS compared to formulations containing 0.5 mg/ml PS20 after 7 days of shaking at 25°C. Compared with the materials, a slight increase in the content of HMWS and charge variants was observed (Fig. 11). Therefore, a formulation containing 0.5 mg/ml PS20 was selected. A polysorbate 20 level of 0.5 mg/ml was shown to be sufficient to protect geffirumab from stresses that may occur during processing (e.g., stirring, freezing and thawing, or shear stress), handling, storage, and transportation. The concentration range of 0.2-0.8 mg/ml PS20 was further evaluated in a subsequent multivariable formulation robustness study (see Example 5, Formulation Robustness Study). This study confirmed acceptable stability behavior within this concentration range. Example 5 : Formulation Robustness Study

The composition of drug substances and drug products can vary within certain limits based on manufacturing factors such as weighing tolerances of buffer components. A multivariable formulation robustness study was performed and it demonstrated that the relevant quality attributes (QA) of geffirumab were acceptable at the edge of the range of these compositions. A two-level multivariate stability study was conducted on three factors that have been identified as having a potential impact on critical quality attributes (CQAs) of pharmaceutical products during storage. The following three formulation parameters were evaluated: 1. Protein concentration 2. pH 3.PS20 concentration In addition, three formulation parameters were separately evaluated in a univariate stability study: 4. Buffering strength 5. L-methionine concentration 6.D-sucrose concentration

Multivariable formulation robustness studies demonstrated that the associated CQAs for gaffetuzumab were acceptable across the entire range of required formulation compositions. research design

Risk assessments are performed to identify pharmaceutical substances and formulation parameters in pharmaceutical products that are important to maintain product quality during the shelf life. Multivariate and univariate studies have been set up accordingly.

multivariate study (F6 to F12)

A partial factorial design (Resolution III) stability study was performed at two levels using the three identified formulation parameters protein concentration, pH, and PS20 concentration as input factors.

Univariate study (F13 to F20)

L-methionine and D-sucrose concentrations (low and high levels) and buffering strengths (low and high levels) were tested.

A formulation with low protein concentration, low pH, and low PS20 concentration was evaluated in direct comparison with the corresponding formulation at high pH, high protein concentration, and high PS20 concentration.

One formulation with a PS20 concentration of 0.3 mg/ml was included to support the acceptance standard setting.

The formulation parameter ranges tested were defined to cover the drug product specification acceptance criteria and/or manufacturing acceptable ranges, as described in Table 5. Table 6 shows a design consisting of 15 experiments with 3 center points corresponding to the target commercial formulation compositions. Table 5 : Formulation Robustness Study: Target Formulation and Multivariate and Univariate Study Scope target lower content higher content Gerfituzumab concentration (mg/ml) Mab 1 0.9 1.1 L-histamine/L-histamine hydrochloride (mM) His 20 15 25 pH pH 5.5 5.0 6.0 PS20 concentration (mg/ml) PS20 0.5 0.2 (0.3) 0.8 D-sucrose concentration (mM) Suc 240 200 280 L-methionine concentration (mM) Met 10 5 15 Table 6 : Formulation Robustness Study Design: Gaffetuzumab Formulation Evaluated multivariate study Tested in univariate studies Preparations Protein concentration (mg/ml) pH PS20 concentration (mg/ml) Buffer strength (mM) D-sucrose concentration (mM) L-methionine concentration (mM) F6 0.90 5.0 0.80 20 240 10 F7 1.10 5.0 0.20 F8 0.90 6.0 0.20 F9 1.10 6.0 0.80 F10 (target) 1.00 5.5 0.50 F11 (target) 1.00 5.5 0.50 F12 (target) 1.00 5.5 0.50 Univariate study F13 0.90 5.0 0.20 20 240 10 F14 1.00 5.5 0.50 20 200 10 F15 1.00 5.5 0.50 20 280 10 F16 1.00 5.5 0.50 20 240 5 F17 1.00 5.5 0.50 20 240 15 F18 1.00 5.5 0.50 15 240 10 F19 1.00 5.5 0.50 25 240 10 F20 1.00 5.5 0.30 20 240 10 The stability of gaffetuzumab in the formulation compositions described in Table 6 was evaluated as: l Stability studies: o Storage conditions: real-time (2°C-8°C) and accelerated (25°C) o Testing frequency: at Stored under the above storage conditions for 0, 4, 13, 26 (end of storage at 25℃), 39, 52, 78 and 104 weeks l Stress test: o 5 freeze-thaw cycles, o 2-8℃ shaking for one week and 25℃ shaking for one week Supporting the stability of DS: QA evaluated at 0, 26, 52 and 104 weeks of storage at -40°C: o HMWS (high molecular weight species) and the main peak by SE-HPLC o LMWS (low molecular weight species) and by non-reduction The main peak of CE-SDS, o Acidic peaks 2 and 3, acidic zone, basic zone and main peak by IE-HPLC o Protein content by UV-visible spectroscopy o Polysorbate 20 content by HPLC-ELSD o Borrow L-methionine and L-histidine concentration by RP-HPLC o Oxidation and isomerization by peptide mapping (LC-MS) o Potency by bioassay o Visible particles o Invisible particles by visual inspection o Color, clarity/opalescence o pH o Osmolarity o Density profile analysis procedure

Data on all quality attributes were collected for each blend over time. The relative change over time for each QA was assessed. Multivariate studies:

A simple linear regression fit was performed for each quality attribute and for each formulation over time. Therefore, the degradation rate was calculated for each quality attribute and for each formulation. If not explicitly mentioned, degradation rates are reported as weekly degradation. These degradation rates were evaluated as responses in a design of experiments (DoE) study, and the effect of three parameters: protein concentration, pH, and PS20 concentration on these degradations was investigated. If the quality attributes do not show meaningful changes over time compared to the target formulation, regression analysis and effect estimation will not be performed. For quality attributes that showed meaningful changes over time, linear regression was used to estimate the main effects of the three factors on the degradation rate. Additionally, main renderings are shown to illustrate these effects graphically. Univariate study:

For parameters tested in the univariate study, results after 39 weeks of storage at 2°C-8°C were evaluated compared to T0 to identify potential changes. If a change is identified, the degradation rate is calculated and compared to the degradation of the target formulation to estimate the marginal impact of the investigated formulation parameters. In some cases, weekly degradation rates were multiplied by a factor of 104 to convert to observed degradation rates over 104 weeks. Regression analyzes were performed using JMP® software (SAS Institute, Cary, NC, version 10.0 or later). Robust formulation stability under recommended storage conditions (2°C-8°C) :

Table 7 provides an overview of the evaluation of relative changes compared to the target formulation after 39 weeks of storage at 2°C-8°C. Increased acidic variant content (by acidic zone and acidic peak 2 of IE-HPLC) was observed for all formulations formulated at pH 6 (F8, F9, F20). The observed increase in acidic variants is reflected by a corresponding decrease in the main IE-HPLC peak in the affected formulations. No changes were observed in all other CQAs for all other formulations after 39 weeks of storage at 2°C-8°C. Overall, pH was identified as a critical formulation parameter. All other formulation parameters (protein content, PS20, L-methionine and D-sucrose concentrations, and buffer strength) were not shown to have an impact on test CQAs within the scope of the investigation. Stability of robust formulations under accelerated storage conditions (25°C) :

Compared to the 2°C-8°C data, an increase due to deamidation (acidic zone and acidic peak 2 by IE-HPLC) was observed for all formulations formulated at pH 6 (F8, F9, F20) Acidic variant content, which is reflected by a decrease in the main IE-HPLC peak in the affected formulations. Additionally, for F1 and F2 formulated at pH 5, an increase in fragment content was observed by an increase in LMWS by CE-SDS. This increase is reflected in the decrease in the main peak of CE-SDS. No changes were observed in any other CQA for all other formulations after 26 weeks of storage at 25°C.

Overall, the 25°C data confirm that pH is a critical formulation parameter. All other formulation parameters showed no impact on CQA. Table 7 : Relative changes in relevant CQAs after 39 weeks of storage at 2℃-8℃ Relative change from target formulation Description compared to target formulation Purity by SE-HPLC Sum of HMW no change main peak no change By the purity of NR-CE-SDS Sum of LMWS no change main peak no change Purity by IE-HPLC Acidic peak 2 Increase All formulations increased at pH 6 (F8, F9, F20) Acidic peak 3 no change acidic zone Increase All formulations increased at pH 6 (F8, F9, F20) main peak reduce All formulations decreased at pH 6 (F8, F9, F20) protein concentration no change Polysorbate 20 Concentration no change L-Methionine and L-Histidine Concentrations by RP-HPLC ^a NA Tryptophan and methionine oxidation no change Aspartic acid ^{isomerizationb} no change By the power of biometrics no change Visible particles no change Invisible particles no change Color, Clarity/Opalescence no change Solution pH no change ^Osmolaritya NA ^{aMeasurements} at t=0 and end of study 104 weeks later. ^b Only measured at t=0 after 52 weeks and 104 weeks of storage at 2℃-8℃. Robust formulation stability under recommended storage conditions for drug substance (-40°C) :

To support the stability of the drug substance over the entire range of required formulation compositions, stability studies were performed on drug product robustness formulations stored at -40°C. The results confirmed that no significant changes in the tested quality attributes were observed when the formulations were stored at the recommended storage conditions for pharmaceutical substances at -40°C for 26 weeks. Robust formulation stability after shaking and freeze / thaw stress:

The formulations were shaken at 2°C-8°C or 25°C for one week. Additionally, the formulations were evaluated after five freeze/thaw cycles between -40°C and 5°C. All samples contained few visible particles under shaking or freezing/thawing stress.

Visually invisible particles were unchanged for all formulations under shaking and freeze/thaw stress. Formulations with low PS20 content (0.2 mg/ml, F7, F8, F13) did not show any product quality after shaking and freeze/thaw stress compared to all other formulations containing 0.3-0.8 mg/ml PS20 content influence.

This result confirms that polysorbate 20 content of ≥0.2 mg/ml is sufficient to protect proteins from shaking and freeze/thaw stress. In contrast, the formulation with low D-sucrose content (200 mM, F19) did not show any product after shaking and freeze/thaw stress compared to all other formulations containing 240-280 mM D-sucrose content quality impact. This result confirms that ≥200 mM D-sucrose content is sufficient to protect proteins from freezing/thawing stress. No substantial changes in any other quality attributes were observed under shaking or freeze/thaw stress when compared to control samples. Linear regression analysis of identified CQAs based on data under recommended storage conditions (2°C-8°C) :

A simple linear regression analysis was performed on the affected CQAs: solution pH, protein concentration and PS20 concentration. pH was identified as having a major effect. The calculated weekly degradation rate is extrapolated to the end of shelf life (EoS) by multiplying by 104 weeks (= 24 months). The inferred results are summarized in Table 8.

Linear regression analysis showed that the pH range tested had no meaningful impact on the identified CQAs, as all CQAs were within the stability acceptance criteria. However, in order to control the increase in acidic zones, the pH acceptance criteria for drug product release are limited to 5.2-5.8. Table 8 : Linear regression analysis results based on 2℃-8℃ data CQA pH 5 ^a Inferred degradation rate after 104 weeks pH 5 ^a Calculated ^value for EoSb pH 6 ^a Inferred degradation rate after 104 weeks ^Calculated for pH 6 ^a EoSb Target (pH 5.5) Inferred degradation rate after 104 weeks Calculated ^value of target (pH 5.5) EoSb Acidic peak 2 (area %) 0.298 5.9 1.829 7.4 1.064 6.7 Acidic peak 3 (area %) 0.147 2.8 0.481 3.1 0.314 2.9 Acidic zone (area %) 1.509 15.7 3.996 18.2 2.753 16.9 LMWS (area%) 0.574 2.4 0.134 2.0 0.354 2.2 ^aAll other parameters were set as targets for linear regression analysis. ^b Calculated as t = 0 + degradation rate after 104 weeks (using average t = 0 for all formulations). Conclusion:

Extrapolated data indicate that a high pH of 6.0 has an effect on the content of acidic variants after 24 months (the required shelf life of the drug product). Therefore, the pH acceptance criteria for drug product release are limited to 5.2-5.8 to limit the formation of acidic forms during drug product stability. Formulations are considered robust until end of shelf life because: l For all formulations at the edge of the formulation range, the CQA meets release acceptance criteria at t = 0 and after 9 months of storage at 2°C-8°Cl The CQA meets the stability acceptance criteria when extrapolating the EoS for all formulations to the edge of the formulation range using degradation rates. Example 6 : Evaluation of Polysorbate 20 Degradation

Polysorbate 20 can degrade via oxidative or hydrolytic mechanisms. Hydrolytic degradation of polysorbate 20 results in the formation of free fatty acids (FFA) such as lauric acid. At certain high concentrations, FFA may form visible or invisible particles. Additionally, polysorbate 20 degradation can be a concern if it results in the formulation containing less polysorbate than required to protect the protein from agitation stress.

Because of these issues, polysorbate 20 degradation was monitored during formulation development. PS20 degradation in gaffetuzumab formulations was observed during formulation development to be dependent on protein concentration. For the 25 mg/ml formulation (Figure 12), visible particles were observed at 2°C-8°C and significant PS20 degradation was observed. For the 5 mg/ml formulation, PS20 degradation was less pronounced and visible particles were observed after 20 months. Invisible particle counts were not affected. Visible particles were isolated and characterized by Fourier transform infrared (FTIR) analysis and found to be FFA. The 1 mg/ml formulation showed no PS20 degradation (exceeding method precision) and no visible particle formation over the 24-month study period. Invisible particle counts are always low. Long-term stability data for nine drug product (DP) batches from four different drug substance (DS) batches confirmed the absence of visible particles. Figure 13 provides a visualization of the long-term stability data for an example DP batch. Example 7 : Stability studies in physical and chemical applications

The drug product Gerfituzumab is supplied as a sterile liquid concentrate for IV infusion solution. The drug product consists of 1 mg/ml gratinumab, 240 mM sucrose, 10 mM L-methionine, 0.5 mg/ml in 20 mM L-histamine/L-histamine hydrochloride buffer. Composed of polysorbate 20, pH 5.5. Gerfituzumab is a preservative-free drug product available in single-dose 2.5-ml and 10-ml glass vials. Gerfituzumab is intended for IV administration via IV bag infusion upon dilution in 0.9% or 0.45% sodium chloride. The recommended registered dose and schedule based on an ascending dosing schedule is 2.5/10/30 mg. With dosages of 0.05 mg/ml to 0.6 mg/ml solution concentration in IV bag depends on dose. In a group approach, 0.05 mg/ml, 0.1 mg/ml, and 0.6 mg/ml dose solutions were tested for compatibility to cover the entire dose range (Table 9).

Stability and compatibility studies are performed to confirm the physicochemical stability of solutions for infusion under recommended conditions of use. Studies have shown that gaffetuzumab solutions for infusion are stable under typical preparation and administration procedures at 2°C-8°C for 72 hours and at 30°C under ambient room lighting conditions for an additional 24 hours. hours, followed by infusion at ≤ 25°C for no more than 16 hours. The nominal protein concentration range shown to be stable for solutions for infusion is 0.05 to 0.6 mg/ml. Research materials and settings:

The physicochemical stability of gaffetuzumab was evaluated after dilution into 100 ml or 250 ml IV bags containing 0.9% sodium chloride solution and 0.45% sodium chloride solution, simulating handling procedures used in commercial settings. For each diluent, dilution concentrations were evaluated at approximately 0.05 mg/ml (low dose, tested only in 0.9% sodium chloride), 0.1 mg/ml (low dose), and 0.6 mg/ml (high dose) The product quality of gaffetuzumab, including the expected concentration range of the product as summarized in Table 9,

For 0.9% sodium chloride, two different types of bags with drug product contact surfaces made of polyvinyl chloride (PVC) or polyolefin polyethylene polypropylene (PO‑PE‑PP) were tested. For 0.45% sodium chloride, bags with drug product contact surfaces made of PVC were tested. For each diluent, three drug product batches were used for stability evaluation in a matrix setup. Drug product batches have been stored for 20 months, or 7 months at 2°C-8°C. Table 9 : Study setup in simulated use ( 0.9% sodium chloride solution in PVC or PO‑PE‑PP IV bag and 0.45% sodium chloride solution in PVC IV bag ) dose Nominal protein concentration in bag after dilution Remove 0.9% NaCl/ 0.45% NaCl from bag Drug product injected into bag hold time Infusion volume infusion rate low dose 2.5 mg 0.05mg/ml 12.5ml* 12.5ml 2℃-8℃: 72 h 30℃: 24 h 250ml 0.3 mg/h 6 ml/h low dose 2.5 mg 0.1mg/ml 10ml 10ml 2℃-8℃: 72 h 30℃: 24 h 100ml 0.3 mg/h 3 ml/h high dose 30 mg 0.6mg/ml 60ml 60ml 2℃-8℃: 72 h 30℃: 24 h 100ml 0.72 mg/h 1.2 ml/h *Tested in 0.9% NaCl only. Simulate infusion of dosing solution by passing dilute gratinumab solution through: 1. With PVC, polyethylene (PE), polybutadiene (PBD), polyurethane ( Infusion set for product contact surfaces of PUR), silicone and acrylonitrile butadiene styrene (ABS), with or without a 0.2 µm in-line filter made of polystyrene or polyetherstyrene (PES). 2. Three-way stopcock infusion aid made of polycarbonate (PC). 3. Catheters made of polyether polyurethane (PEU) or polytetrafluoroethylene (PTFE)

Simulated infusions were performed over 16 hours, longer than the expected 4-8 hour infusion duration, to ensure compatibility of the dosing solution during prolonged contact with the materials of construction of the infusion set and adjunctive device.

Samples were collected from each IV bag for analysis after dilution and cumulative hold time, and at the end of the simulated infusion.

Test samples using appropriate stability-indicating methods, including purity by SE-HPLC, IE-HPLC and CE-SDS, protein content by UV, visually invisible particles by light shielding, color, transparency/opalescence , pH and efficacy by bioassay. LMW by CE‑SDS was measured only for high doses (0.6 mg/ml) because at sample concentrations ≤ 0.1 mg/ml the signal intensity was too low to meaningfully interpret the data. However, the potency information provided ensures product quality. result:

Usage studies have shown that after dilution into 0.9% or 0.45% sodium chloride solution, gaffetuzumab was maintained at 2°C-8°C for 72 hours, and then maintained at 30°C under ambient room lighting conditions for an additional 24 hours. Hours are physicochemically stable, followed by simulated infusion at ≤ 25°C for no more than 16 hours. For 0.5 mg/ml dose solutions, in-line filters should not be used.

The drug product batches used in these compatibility studies had previously been stored at recommended storage temperatures (2°C-8°C) for 7-20 months, indicating that drug product aging does not affect stability during handling and administration. sex. Example 8 : Microbial Stability

Drug products must be diluted using sterile technique before administration. Prepare gaffetuzumab solutions for IV administration by diluting the drug product into an infusion bag containing 0.9% sodium chloride or 0.45% sodium chloride. The prepared infusion solution should be used immediately. The pharmaceutical product does not contain any antimicrobial preservatives; therefore, the sterility of the solution must be ensured by maintaining appropriate sterile conditions during use.

Microbial challenge studies are performed to evaluate the propensity of a solution to support microbial proliferation to prevent accidental contamination. The proliferation of seven different test microorganisms (listed in USP <51>) was evaluated at 2°C-8°C for up to 96 hours and at 20°C-25°C for up to 48 hours. The results meet the acceptance criterion of "no growth" when the measured difference does not exceed 0.5 log ₁₀ units from the initial value. Other embodiments

Although the above invention has been described in detail by way of illustrations and examples for the purpose of clear understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are expressly incorporated by reference in their entirety.

This application document contains at least one color drawing. Copies of this patent or patent application with color drawing(s) will be provided by the Patent Office upon request and the necessary fee. Figures 1A - 1N : Schematic diagrams showing configurations of exemplary anti- CD20 /anti-CD3 bispecific antibodies. Figure 2 : Schematic diagram showing the structure of gaffetuzumab. Figure 3 : Formulations developed for GLP toxicity and human entry studies. Surfactant content of formulations F1 to F5 initially versus after 6 weeks of storage at 5°C, 25°C or 40°C. Figure 4A to Figure 4C : Formulation development GLP toxicity and human entry studies, particle size screening chromatography (SEC) of formulations F1 to F5, initially and after 6 weeks of storage at 5°C, 25°C, or 40°C. Figure 4A: main peak, Figure 4B: high molecular weight (HMW); Figure 4C: low molecular weight (LMW). Figure 5A to Figure 5C : Formulation development GLP toxicity and human entry studies, ion exchange chromatography (IEC) of formulations F1 to F5, initially and after 6 weeks of storage at 5°C, 25°C or 40°C. Figure 5A: Main peak, Figure 5B. HMW; Figure 5C. LMW. Figure 6 : Formulation Development - Analytical results for Formulation F1 up to 84 weeks. F1 = 5 mg/ml RO7022859 (i.e., gaffetuzumab), 20 mM histidine HCl pH 5.5, 240 mM sucrose, 10 mM methionine, 0.05% (w/v) polysorbate 20. Figure 7A to Figure 7B : Formulation development GLP toxicity and human entry studies, huCD20 binding of formulations F1 to F5, initially and after 3 weeks and 6 weeks of storage at 5°C, 25°C, or 40°C (Figure 7A) and formulations Comparison of huCD3 binding of F1 to F5 initially and after storage at 5°C, 25°C, or 40°C for 3 weeks and 6 weeks (Fig. 7B). Figure 8A - 8B : Phase III and commercial formulation development studies. Size analysis (SE)-HPLC % HMWS (Figure 8A) and ion exchange (IE)-HPLC % acidic zone (Figure 8B) of gaffetuzumab as a function of protein concentration after 104 weeks of storage at 5°C. Figure 9A - 9B : Phase III and commercial formulation development studies. SE-HPLC of geffirumab % HMWS (Figure 9A) and % acidic zone (Figure 9B) as a function of pH and stabilizer (methionine) addition after 6 weeks of storage at 40°C. Figure 10A - 10B : Phase III and commercial formulation development studies. SE-HPLC % HMWS and IE-HPLC % acidic zone of gaffetumumab including visible particle formation as a function of tonicity agent after 26 weeks of storage at 25°C. Figure 11A - 11B : Phase III and commercial formulation development studies. Geffirumab SE-HPLC % HMWS, including visible particle formation (Figure 11A) and IE-HPLC % acidic zone (Figure 11B) as a function of surfactant after 7 days of shaking at 25°C. Figure 12 : Phase III and commercial formulation development studies. Gerfitolumab PS20 content [mg/ml] and visible particle formation as a function of protein concentration initially and after 104 weeks of storage at 5°C. Figure 13 : Long-term stability data: PS20 content for example geffirumab DP batch stability (stored at 2-8°C).

TW202404637A_112113568_SEQL.xml

Claims (32)

A liquid pharmaceutical composition containing: Approximately 1 to 25 mg/ml of anti-CD20/anti-CD3 bispecific antibody; About 10 to 50 mM buffer; About ≥200 mM tonicity agent; approximately 0 to 15 mM methionine; and Approximately ≥ 0.2 mg/ml surfactant; pH in the range of about 5.0 to about 6.0, wherein the anti-CD20/anti-CD3 bispecific antibody contains a) At least one antigen-binding domain that specifically binds to CD20, which includes Heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 6; and b) At least one antigen-binding domain that specifically binds to CD3, which includes Heavy chain variable region, which contains: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising: (i) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 14. The liquid pharmaceutical composition of claim 1, wherein the anti-CD20/anti-CD3 bispecific antibody concentration is in the range of about 1 to 5 mg/ml. The liquid pharmaceutical composition of claim 1 or 2, wherein the concentration of the anti-CD20/anti-CD3 bispecific antibody is in the range of about 0.9 to 1.1 mg/ml. The liquid pharmaceutical composition of any one of claims 1 to 3, wherein the concentration of the anti-CD20/anti-CD3 bispecific antibody is about 1 mg/ml. The liquid pharmaceutical composition of any one of claims 1 to 4, wherein the anti-CD20/anti-CD3 bispecific antibody comprises a) At least one antigen-binding domain that specifically binds to CD20, which includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, and b) At least one antigen-binding domain that specifically binds to CD3, which includes the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16. The liquid pharmaceutical composition of any one of claims 1 to 5, wherein the anti-CD20/anti-CD3 bispecific antibody comprises a) a first Fab molecule that specifically binds to CD3, in particular CD3 epsilon; and in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain replace each other; b) The second Fab and the third Fab molecule, which specifically bind to CD20, wherein in the constant domain CL of the second Fab and the third Fab molecule, the amino acid at position 124 is replaced by lysine (K) is substituted (according to Kabat numbering), and the amino acid at position 123 is substituted (according to Kabat numbering) by lysine (K) or arginine (R), specifically arginine (R), and where In the constant domain CH1 of the second and third Fab molecules, the amino acid at position 147 is replaced by glutamic acid (E) (EU numbering), and the amino acid at position 213 is replaced by glutamic acid (E) supersedes (EU number); and c) Fc domain, which consists of the first unit and the second unit that can stably associate. The liquid pharmaceutical composition of any one of claims 1 to 6, wherein the anti-CD20/anti-CD3 bispecific antibody is glofitumab. For example, the liquid pharmaceutical composition according to any one of claims 1 to 7, wherein the buffer is a histidine buffer, optionally a histidine HCl buffer. The liquid pharmaceutical composition of any one of claims 1 to 8, wherein the buffer is at a concentration of about 15 to 25 mM. The liquid pharmaceutical composition of any one of claims 1 to 9, wherein the buffer is at a concentration of about 20 mM. The liquid pharmaceutical composition of any one of claims 1 to 10, wherein the buffer provides a pH of about 5.2 to about 5.8. The liquid pharmaceutical composition of any one of claims 1 to 11, wherein the tonicity agent is selected from the group of salts, sugars and amino acids. For example, the liquid pharmaceutical composition of claim 12, wherein the tonicity agent is sucrose or sodium chloride. The liquid pharmaceutical composition of claim 13, wherein the tonicity agent is sucrose at a concentration of about 200 mM or higher. Such as the liquid pharmaceutical composition of claim 13 or 14, wherein the tonicity agent is sucrose at a concentration of about 200 mM to 280 mM. The liquid pharmaceutical composition of any one of claims 13 to 15, wherein the tonicity agent is sucrose at a concentration of about 240 mM. The liquid pharmaceutical composition of any one of claims 1 to 16, wherein the methionine is at a concentration of about 5 to 15 mM. The liquid pharmaceutical composition of claim 17, wherein the methionine is at a concentration of about 10 mM. The liquid pharmaceutical composition of any one of claims 1 to 18, wherein the surfactant is at a concentration of about 0.2 to 0.8 mg/ml. The liquid pharmaceutical composition according to any one of claims 1 to 19, wherein the surfactant is polysorbate 20 or poloxamer 188. For example, the liquid pharmaceutical composition of claim 20, wherein the surfactant is polysorbate 20 at a concentration of 0.2 to 0.8 mg/ml. Such as the liquid pharmaceutical composition of claim 21, wherein the surfactant is polysorbate 20 at a concentration of about 0.5 mg/ml. For example, the liquid pharmaceutical composition according to any one of claims 1 to 22, which contains: approximately 1 to 5 mg/ml of the anti-CD20/anti-CD3 bispecific antibody; Approximately 15 to 25 mM histamine buffer; Approximately 200 to 280 mM sucrose; approximately 0 to 15 mM methionine; and Approximately 0.2 to 0.8 mg/ml of PS20 pH is about 5 to about 6. For example, the liquid pharmaceutical composition according to any one of claims 1 to 23, which contains: Approximately 1 mg/ml of greffitumab; Approximately 20 mM histamine buffer; Approximately 240 mM sucrose; Approximately 10 mM methionine; and Approximately 0.5 mg/ml of PS20 The pH is about 5.5. The liquid pharmaceutical composition of any one of claims 1 to 24, wherein the molar ratio of the PS20 to the anti-CD20/anti-CD3 bispecific antibody is less than 100. The liquid pharmaceutical composition of claim 25, wherein the molar ratio of PS20 to anti-CD20/anti-CD3 bispecific antibody is between 50 and 100. The liquid pharmaceutical composition of claim 26, wherein the molar ratio of the PS20 to the anti-CD20/anti-CD3 bispecific antibody is about 79. Use of a liquid pharmaceutical composition according to any one of claims 1 to 27 for the preparation of a medicament for the treatment of cell proliferative disorders. Such as the liquid pharmaceutical composition of any one of claims 1 to 27, which is used to treat or delay the progression of cell proliferative disorders in individuals in need. A method of treating or delaying the progression of a cell proliferative disorder in an individual in need thereof, the method comprising administering to the individual an effective amount of a liquid pharmaceutical composition according to any one of claims 1 to 27. The use, liquid pharmaceutical composition or method of any one of claims 28 to 30, wherein the cell proliferative disorder is cancer. The present invention is as described above.